Vectors and yeast strains for protein production

ABSTRACT

Lower eukaryote host cells in which the function of at least one endogenous gene encoding a chaperone protein, such as a Protein Disulphide Isomerase (PDI), has been reduced or eliminated and at least one mammalian homolog of the chaperone protein is expressed are described. In particular aspects, the host cells further include a deletion or disruption of one or more O-protein mannosyltransferase genes, and/or overexpression of an endogenous or exogenous Ca2 +  ATPase. These host cells are useful for producing recombinant glycoproteins in large amounts and for producing recombinant glycoproteins that have reduced O-glycosylation.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to use of chaperone genes to improveprotein production in recombinant expression systems. In general,recombinant lower eukaryote host cells comprise a nucleic acid encodinga heterologous chaperone protein and a deletion or disruption of thegene encoding the endogenous chaperone protein. These host cells areuseful for producing recombinant glycoproteins in large amounts and forproducing recombinant glycoproteins that have reduced O-glycosylation.

(2) Description of Related Art

Molecular chaperones play a critical role in the folding and secretionof proteins, and in particular, for the folding and secretion ofantibodies. In lower eukaryotes, and particularly in yeast, ProteinDisulfide Isomerase (PDI) is a chaperone protein, which functions tohelp create the disulphide bonds between multimeric proteins, such asthose between antibody heavy and light chains. There have been pastattempts to increase antibody expression levels in P. pastoris byoverexpressing human PDI chaperone protein and/or overexpressingendogenous PDI. See for example, Wittrup et al., U.S. Pat. No.5,772,245; Toyoshima et al., U.S. Pat. Nos. 5,700,678 and 5,874,247; Nget al., U.S. Application Publication No. 2002/0068325; Toman et al., J.Biol. Chem. 275: 23303-23309 (2000); Keizer-Gunnink et al., Martix Biol.19: 29-36 (2000); Vad et al., J. Biotechnol. 116: 251-260 (2005); Inanaet al., Biotechnol. Bioengineer. 93: 771-778 (2005); Zhang et al.,Biotechnol. Prog. 22: 1090-1095 (2006); Damasceno et al., Appl.Microbiol. Biotechnol. 74: 381-389 (2006); and, Huo et al., Proteinexpress. Purif. 54: 234-239 (2007).

Protein disulfide isomerase (PDI) can produce a substantial increase ora substantial decrease in the recovery of disulfide-containing proteins,when compared with the uncatalyzed reaction; a high concentration of PDIin the endoplasmic reticulum (ER) is essential for the expression ofdisulfide-containing proteins (Puig and Gilbert, 1. Biol. Chem.,269:7764-7771 (1994)). The action of PDI1 and its co-chaperones is shownin FIG. 2.

In Gunther et al., J. Biol. Chem., 268:7728-7732 (1993) the Trg1/Pdi1gene of Saccharomyces cerevisiae was replaced by a murine gene of theprotein disulfide isomerase family. It was found that two unglycosylatedmammalian proteins PDI and ERp72 were capable of replacing at least someof the critical functions of Trg1, even though the three proteinsdiverged considerably in the sequences surrounding thethioredoxin-related domains; whereas ERp61 was inactive.

Development of further protein expression systems for yeasts andfilamentous fungi, such as Pichia pastoris, based on improved vectorsand host cell lines in which effective chaperone proteins wouldfacilitate development of genetically enhanced yeast strains for therecombinant production of proteins, and in particular, for recombinantproduction of antibodies.

The present invention provides improved methods and materials for theproduction of recombinant proteins using auxiliary genes and chaperoneproteins. In one embodiment, genetic engineering to humanize thechaperone pathway resulted in improved yield of recombinant antibodyproduced in Pichia pastoris cells.

As described herein, there are many attributes of the methods andmaterials of the present invention which provide unobvious advantagesfor such expression processes over prior known expression processes.

BRIEF SUMMARY OF THE INVENTION

The present inventors have found that expression of recombinant proteinsin a recombinant host cell can be improved by replacing one or more ofthe endogenous chaperone proteins in the recombinant host cell with oneor more heterologous chaperone proteins. In general, it has been foundthat expression of a recombinant protein can be increased when the geneencoding an endogenous chaperone protein is replaced with a heterologousgene from the same or similar species as that of the recombinant proteinto be produced in the host cell encoding a homolog of the endogenouschaperone protein. For example, the function of an endogenous geneencoding a chaperone protein can be reduced or eliminated in a lowereukaryotic host cell and a heterologous gene encoding a mammalianchaperone protein is introduced into the host cell. In general, themammalian chaperone is selected to be from the same species as therecombinant protein that is to be produced by the host cell. The lowereukaryotic host cell that expresses the mammalian chaperone protein butnot its endogenous chaperone protein is able to produce active,correctly folded recombinant proteins in high amounts. This is animprovement in productivity compared to production of the recombinantprotein in lower eukaryotic host cells that retain the endogenous PDIgene.

The present inventors have also found that by improving proteinexpression as described herein provides the further advantage thathealthy, viable recombinant host cells that have a deletion ordisruption of one or more of its endogenous proteinO-mannosyltransferases (PMT) genes can be constructed. Deleting ordisrupting one or more of the PMT genes in a lower eukaryotic cellresults in a reduction in the amount of O-glycosylation of recombinantproteins produced in the cell. However, when PMT deletions are made inlower eukaryotic host cells that further include a deletion in one orgenes encoding mannosyltransferases and express the endogenous chaperoneproteins, the resulting cells often proved to be non-viable orlow-producing cells, rendering them inappropriate for commercial use.

Thus, in certain aspects, the present invention provides lowereukaryotic host cells, in which the function of at least one endogenousgene encoding a chaperone protein has been reduced or eliminated, and anucleic acid molecule encoding at least one mammalian homolog of thechaperone protein is expressed in the host cell. In further aspects, thelower eukaryotic host cell is a yeast or filamentous fungi host cell.

In further still aspects, the function of the endogeneous gene encodingthe chaperone protein Protein Disulphide Isomerase (PDI) is disrupted ordeleted such that the endogenous PDI1 is no longer present in the hostcell and a nucleic acid molecule encoding a mammalian PDI protein isintroduced into the host cell and expressed in the host cell. In oneembodiment, the mammalian PDI protein is of the same species as that ofthe recombinant proteins to be expressed in the host cell and that thenucleic acid molecule encoding the mammalian PDI be integrated into thegenome of the host cell. For example, when the recombinant protein isexpressed from a human gene introduced into the host cell, it ispreferable that the gene encoding the PDI be of human origin as well. Infurther embodiments, the nucleic acid molecule for expressing the PDIcomprises regulatory elements, such as promoter and transcriptiontermination sequences, which are functional in the host cell, operablylinked to an open reading frame encoding the mammalian PDI protein. Inother embodiments, the endogenous PDI gene is replaced with a nucleicacid molecule encoding a mammalian PDI gene. This can be accomplished byhomologous recombination or a single substitution event in which theendogenous PDI1 gene is looped out by the mammalian PDI gene, comprisingoverlapping sequences on both ends.

In further aspects, the lower eukaryotic host cells of the invention arefurther transformed with a recombinant vector comprising regulatorynucleotide sequences derived from lower eukaryotic host cells and acoding sequence encoding a selected mammalian protein to be produced bythe above host cells. In certain aspects, the selected mammalian proteinis a therapeutic protein, and may be a glycoprotein, such as anantibody.

The present invention also provides lower eukaryotic host cells, such asyeast and filamentous fungal host cells, wherein, in addition toreplacing the genes encoding one or more of the endogenous chaperoneproteins as described above, the function of at least one endogenousgene encoding a protein O-mannosyltransferase (PMT) protein is reduced,disrupted, or deleted. In particular embodiments, the function of atleast one endogenous PMT gene selected from the group consisting of thePMT1 and PMT4 genes is reduced, disrupted, or deleted.

In further embodiments, the host cell may be a yeast or filamentousfungal host cell, such as a Pichia pastoris cell, in which theendogenous Pichia pastoris PDI1 has been replaced with a mammalian PDIand the host cell further expresses a vector comprising regulatorynucleotide sequences derived from or functional in Pichia pastoris cellsoperably linked with an open reading frame encoding a human therapeuticglycoprotein, such as an antibody, which is introduced into the hostcell. The host cell is then further be engineered to reduce or eliminatethe function of at least one endogenous Pichia pastoris gene encoding aprotein O-mannosyltransferase (PMT) protein selected from the groupconsisting of PMT1 and PMT4 to provide a host cell that is capable ofmaking recombinant proteins having reduced O-glycosylation compared tohost cells that have functional PMT genes. In further aspects, the hostcells are further contacted with one or more inhibitors of PMT geneexpression or PMT protein function.

In further aspects, the present invention comprises recombinant hostcells, such as non-human eukaryotic host cells, lower eukaryotic hostcells, and yeast and filamentous fungal host cells, with improvedcharacteristics for production of recombinant glycoproteins,glycoproteins of mammalian origin including human proteins. Therecombinant host cells of the present invention have been modified byreduction or elimination of the function of at least one endogenous geneencoding a chaperone protein. Reduction or elimination of the functionof endogenous genes can be accomplished by any method known in the art,and can be accomplished by alteration of the genetic locus of theendogenous gene, for example, by mutation, insertion or deletion ofgenetic sequences sufficient to reduce or eliminate the function of theendogenous gene. The chaperone proteins whose function may be reduced oreliminated include, but are not limited to, PDI. In one embodiment, theendogenous gene encoding PDI is either deleted or altered in a mannerwhich reduces or eliminates its function.

In further aspects, the function of the chaperone protein is reduced oreliminated and is then replaced, for example, by transforming the hostcell with at least one non-endogenous gene which encodes a homolog ofthe chaperone protein which has been disrupted or deleted. In furtheraspects, the host cells are transformed to express at least one foreigngene encoding a human or mammalian homolog of the chaperone proteinwhich has been disrupted or deleted. In further aspects, the foreigngene encodes a homolog from the same species as, or a species closelyrelated to, the species of origin of the recombinant glycoprotein to beproduced using the host cell.

In particular aspects, the function of the endogenous chaperone proteinPDI1 is reduced or eliminated, and the host cell is transformed toexpress a homolog of PDI which originates from the same species as, or aspecies closely related to, the species of origin of the recombinantprotein to be produced using the host cell. For example, in a Pichiapastoris expression system for expression of mammalian proteins, thePichia pastoris host cell is modified to reduce or eliminate thefunction of the endogenous PDI1 gene, and the host cell is transformedwith a nucleic acid molecule which encodes a mammalian PDI gene.

The present invention also provides methods for increasing theproductivity of recombinant human or mammalian glycoproteins in anon-human eukaryotic host cell, lower eukaryotic host cell, or a yeastor filamentous fungal host cell. The methods of the present inventioncomprise the step of reducing or eliminating the function of at leastone endogenous gene encoding a chaperone protein. Generally, the methodfurther comprises transforming the host cell with at least oneheterogeneous gene which encodes a homolog of the chaperone protein inwhich the function has been reduced or eliminated. The heterogeneousgenes comprise foreign genes encoding human or mammalian homologs of thechaperone proteins in which the functions have been reduced oreliminated. In further aspects, the foreign gene encodes a homolog fromthe same species as, or a species closely related to, the species oforigin of the recombinant glycoprotein to be produced using the hostcell. In many aspects, the chaperone proteins whose function may bereduced or eliminated include PDI.

Thus, further provide are methods for producing a recombinant protein inthe host cells disclosed herein, for example, in one embodiment, themethod comprises providing a lower eukaryotic host cell in which thefunction of at least one endogenous gene encoding a chaperone proteinhas been disrupted or deleted and a nucleic acid molecule encoding atleast one mammalian homolog of the endogenous chaperone protein isexpressed in the host cell: introducing a nucleic acid molecule into thehost cell encoding the recombinant protein: and growing the host cellunder conditions suitable for producing the recombinant protein. Inanother embodiment, the method comprises providing a lower eukaryotichost cell in which the function of (i) at least one endogenous geneencoding a chaperone protein; and (ii) at least one endogenous geneencoding a protein O-mannosyltransferase (PMT) protein; have beenreduced, disrupted, or deleted; and a nucleic acid molecule encoding atleast one mammalian homolog of the chaperone protein is expressed in thehost cell; introducing a nucleic acid molecule into the host cellencoding the recombinant protein: and growing the host cell underconditions suitable for producing the recombinant protein. In anotherembodiment, the method comprises providing lower eukaryotic host cell inwhich the function of the endogenous gene encoding a chaperone proteinPDI; and at least one endogenous gene encoding a proteinO-mannosyltransferase-1 (PMT1) or PMT4 protein; have been reduced,disrupted, or deleted; and a nucleic acid molecule encoding at least onemammalian homolog of the chaperone protein PDI is expressed in the hostcell; introducing a nucleic acid molecule into the host cell encodingthe recombinant protein: and growing the host cell under conditionssuitable for producing the recombinant protein.

It has further been found that overexpressing an Ca²⁺ ATPase in theabove host cells herein effects a decrease in O-glycan occupancy. It hasalso been found that overexpressing a calreticulin and an ERp57 proteinin the above host cells also effected a reduction in O-glycan occupancy.Thus, in further embodiments of the above host cells, the host cellfurther includes one or more nucleic acid molecules encoding one or moreexogenous or endogenous Ca²⁺ ATPases operably linked to a heterologouspromoter. In further embodiments, the Ca²⁺ATPase is the Ca²⁺ ATPaseencoded by the Pichia pastoris PMR1 gene or the Arabidopsis thalianaECAI gene. In further embodiments, the host cells further include one ormore nucleic acid molecules encoding a calreticulin and/or an ERp57.Other Ca²⁺ ATPases that are suitable include but are not limited tohuman SERCA2b protein (ATP2A2 ATPase, Ca⁺⁺ transporting, cardiac muscle,slow twitch 2) and the Pichia pastoris COD1 protein (homologue ofSaccharomyces cerevisiae SPF1). Other proteins that are suitable includebut are not limited to human UGGT (UDP-glucose:glycoproteinglucosyltransferase) protein and human ERp27 protein.

Thus, the present invention provides a lower eukaryote host cell inwhich the function of at least one endogenous gene encoding a chaperoneprotein has been disrupted or deleted and a nucleic acid moleculeencoding at least one mammalian homolog of the endogenous chaperoneprotein is expressed in the host cell.

In a further embodiments, the chaperone protein that is disrupted is aProtein Disulphide Isomerase (PDI) and in further embodiments, themammalian homolog is a human PDI.

In general, the lower eukaryote host cell further includes a nucleicacid molecule encoding a recombinant protein, which in particularaspects, is a glycoprotein, which in further aspects is an antibody orfragment thereof such as Fc or Fab.

In further embodiments, the function of at least one endogenous geneencoding a protein O-mannosyltransferase (PMT) protein has been reduced,disrupted, or deleted. In particular aspects, the PMT protein isselected from the group consisting of PMT1 and PMT4. Thus, the host cellcan further include reduction, disruption, or deletion of the PMT1 orPMT4 alone or reduction, disruption, or deletion of both the PMT1 andPMT4. Thus, further provided is a lower eukaryote host cell in which thefunction of (a) at least one endogenous gene encoding a chaperoneprotein; and (b) at least one endogenous gene encoding a proteinO-mannosyltransferase (PMT) protein; have been reduced, disrupted, ordeleted; and a nucleic acid molecule encoding at least one mammalianhomolog of the chaperone protein is expressed in the host cell.

In further embodiments, the host cell further includes a nucleic acidmolecule encoding an endogenous or heterologous Ca²⁺ ATPase. Inparticular aspects, the Ca²⁺ ATP is selected from the group consistingof the Pichia pastoris PMR1 and the Arabidopsis thaliana ECA1. Thus,further provided is a lower eukaryote host cell in which the function of(a) at least one endogenous gene encoding a chaperone protein has beenreduced, disrupted, or deleted; and nucleic acid molecules encoding atleast one mammalian homolog of the chaperone protein and at least oneCa²⁺ ATPase are expressed in the host cell. Further provided is a lowereukaryote host cell in which the function of (a) at least one endogenousgene encoding a chaperone protein; and (b) at least one endogenous geneencoding a protein O-mannosyltransferase (PMT) protein; have beenreduced, disrupted, or deleted; and nucleic acid molecules encoding atleast one mammalian homolog of the chaperone protein and at least oneCa²⁺ ATPase are expressed in the host cell.

In further still aspects, the host cell further includes a nucleic acidmolecule encoding the human ERp57 chaparone protein or a nucleic acidmolecule encoding a calreticulin (CRT) protein, or both. In particularaspects, the calreticulin protein is the human CRT and the ERp57 is thehuman ERp57. Thus, further provided is a lower eukaryote host cell inwhich the function of (a) at least one endogenous gene encoding achaperone protein has been reduced, disrupted, or deleted; and nucleicacid molecules encoding at least one mammalian homolog of the chaperoneprotein and at least one of CRT or ERp57 are expressed in the host cell.Further provided is a lower eukaryote host cell in which the function of(a) at least one endogenous gene encoding a chaperone protein has beenreduced, disrupted, or deleted; and nucleic acid molecules encoding atleast one mammalian homolog of the chaperone protein, at least one ofCRT or ERp57, and at least one Ca²⁺ ATPase are expressed in the hostcell. Further provided is a lower eukaryote host cell in which thefunction of (a) at least one endogenous gene encoding a chaperoneprotein; and (b) at least one endogenous gene encoding a proteinO-mannosyltransferase (PMT) protein; have been reduced, disrupted, ordeleted; and nucleic acid molecules encoding at least one mammalianhomolog of the chaperone protein, at least one of CRT or ERp57, and atleast one Ca²⁺ ATPase are expressed in the host cell.

In further aspects of the above host cells, the host cell is selectedfrom the group consisting of Pichia pastoris, Pichia finlandica, Pichiatrehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta(Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stipitis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Schizosacchromyces pombe,Schizosacchroyces sp. Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum,Physcomitrella patens and Neurospora crassa. Pichia sp., anySaccharomyces sp., any Schizosacchroyces sp., Hansenula polymorpha, anyKluyveromyces sp., Candida albicans, any Aspergillus sp., Trichodermareesei, Chrysosporium lucknowense, any Fusarium sp. and Neurosporacrass.

Further embodiments include methods for producing recombinant proteinsin yields higher than is obtainable in host cells that are not modifiedas disclosed herein and for producing recombinant proteins that havereduced O-glycosylation or O-glycan occupancy compared to recombinantglycoproteins that do not include the genetic modifications disclosedherein. Recombinant proteins include proteins and glycoproteins oftherapeutic relevance, including antibodies and fragments thereof.

Thus, provided is a method for producing a recombinant proteincomprising: (a) providing a lower eukaryote host cell in which thefunction of at least one endogenous gene encoding a chaperone proteinhas been disrupted or deleted and a nucleic acid molecule encoding atleast one mammalian homolog of the endogenous chaperone protein isexpressed in the host cell; (b) introducing a nucleic acid molecule intothe host cell encoding the recombinant protein: and (c) growing the hostcell under conditions suitable for producing the recombinant protein.

Further provided is a method for producing a recombinant proteincomprising: (a) providing a lower eukaryote host cell in which thefunction of (a) at least one endogenous gene encoding a chaperoneprotein; and (b) at least one endogenous gene encoding a proteinO-mannosyltransferase (PMT) protein; have been reduced, disrupted, ordeleted; and a nucleic acid molecule encoding at least one mammalianhomolog of the chaperone protein is expressed in the host cell; (b)introducing a nucleic acid molecule into the host cell encoding therecombinant protein: and (c) growing the host cell under conditionssuitable for producing the recombinant protein.

Further provided is a method for producing a recombinant proteincomprising: (a) providing a lower eukaryote host cell in which thefunction of (a) at least one endogenous gene encoding a chaperoneprotein; and (b) at least one endogenous gene encoding a proteinO-mannosyltransferase (PMT) protein; have been reduced, disrupted, ordeleted; and nucleic acid molecules encoding at least one mammalianhomolog of the chaperone protein and at least one Ca²⁺ ATPase areexpressed in the host cell; (b) introducing a nucleic acid molecule intothe host cell encoding the recombinant protein: and (c) growing the hostcell under conditions suitable for producing the recombinant protein.

Further provided is a method for producing a recombinant proteincomprising: (a) providing a lower eukaryote host cell in which thefunction of (a) at least one endogenous gene encoding a chaperoneprotein has been reduced, disrupted, or deleted; and nucleic acidmolecules encoding at least one mammalian homolog of the chaperoneprotein and at least one of CRT or ERp57 are expressed in the host cell;(b) introducing a nucleic acid molecule into the host cell encoding therecombinant protein: and (c) growing the host cell under conditionssuitable for producing the recombinant protein.

Further provided is a method for producing a recombinant proteincomprising: (a) providing a lower eukaryote host cell in which thefunction of (a) at least one endogenous gene encoding a chaperoneprotein has been reduced, disrupted, or deleted; and nucleic acidmolecules encoding at least one mammalian homolog of the chaperoneprotein, at least one of CRT or ERp57, and at least one Ca²⁺ ATPase areexpressed in the host cell; (b) introducing a nucleic acid molecule intothe host cell encoding the recombinant protein: and (c) growing the hostcell under conditions suitable for producing the recombinant protein.

Further provided is a method for producing a recombinant proteincomprising: (a) providing a lower eukaryote host cell in which thefunction of (a) at least one endogenous gene encoding a chaperoneprotein; and (b) at least one endogenous gene encoding a proteinO-mannosyltransferase (PMT) protein; have been reduced, disrupted, ordeleted; and nucleic acid molecules encoding at least one mammalianhomolog of the chaperone protein, at least one of CRT or ERp57, and atleast one Ca²⁺ ATPase are expressed in the host cell; (b) introducing anucleic acid molecule into the host cell encoding the recombinantprotein: and (c) growing the host cell under conditions suitable forproducing the recombinant protein.

Further provided is a method for producing a recombinant protein withreduced O-glycosylation or O-glycan occupancy comprising: (a) providinga lower eukaryote host cell in which the function of at least oneendogenous gene encoding a chaperone protein has been disrupted ordeleted and a nucleic acid molecule encoding at least one mammalianhomolog of the endogenous chaperone protein is expressed in the hostcell; (b) introducing a nucleic acid molecule into the host cellencoding the recombinant protein: and (c) growing the host cell underconditions suitable for producing the recombinant protein.

Further provided is a method for producing a recombinant protein withreduced O-glycosylation or O-glycan occupancy comprising: (a) providinga lower eukaryote host cell in which the function of (a) at least oneendogenous gene encoding a chaperone protein; and (b) at least oneendogenous gene encoding a protein O-mannosyltransferase (PMT) protein;have been reduced, disrupted, or deleted; and a nucleic acid moleculeencoding at least one mammalian homolog of the chaperone protein isexpressed in the host cell; (b) introducing a nucleic acid molecule intothe host cell encoding the recombinant protein: and (c) growing the hostcell under conditions suitable for producing the recombinant protein.

Further provided is a method for producing a recombinant protein withreduced O-glycosylation or O-glycan occupancy comprising: (a) providinga lower eukaryote host cell in which the function of (a) at least oneendogenous gene encoding a chaperone protein; and (b) at least oneendogenous gene encoding a protein O-mannosyltransferase (PMT) protein;have been reduced, disrupted, or deleted; and nucleic acid moleculesencoding at least one mammalian homolog of the chaperone protein and atleast one Ca²⁺ ATPase are expressed in the host cell; (b) introducing anucleic acid molecule into the host cell encoding the recombinantprotein: and (c) growing the host cell under conditions suitable forproducing the recombinant protein.

Further provided is a method for producing a recombinant protein withreduced O-glycosylation or O-glycan occupancy comprising: (a) providinga lower eukaryote host cell in which the function of (a) at least oneendogenous gene encoding a chaperone protein has been reduced,disrupted, or deleted; and nucleic acid molecules encoding at least onemammalian homolog of the chaperone protein and at least one of CRT orERp57 are expressed in the host cell; (b) introducing a nucleic acidmolecule into the host cell encoding the recombinant protein: and (c)growing the host cell under conditions suitable for producing therecombinant protein.

Further provided is a method for producing a recombinant protein withreduced O-glycosylation or O-glycan occupancy comprising: (a) providinga lower eukaryote host cell in which the function of (a) at least oneendogenous gene encoding a chaperone protein has been reduced,disrupted, or deleted; and nucleic acid molecules encoding at least onemammalian homolog of the chaperone protein, at least one of CRT orERp57, and at least one Ca²⁺ ATPase are expressed in the host cell; (b)introducing a nucleic acid molecule into the host cell encoding therecombinant protein: and (c) growing the host cell under conditionssuitable for producing the recombinant protein.

Further provided is a method for producing a recombinant protein withreduced O-glycosylation or O-glycan occupancy comprising: (a) providinga lower eukaryote host cell in which the function of (a) at least oneendogenous gene encoding a chaperone protein; and (b) at least oneendogenous gene encoding a protein O-mannosyltransferase (PMT) protein;have been reduced, disrupted, or deleted; and nucleic acid moleculesencoding at least one mammalian homolog of the chaperone protein, atleast one of CRT or ERp57, and at least one Ca²⁺ ATPase are expressed inthe host cell; (b) introducing a nucleic acid molecule into the hostcell encoding the recombinant protein: and (c) growing the host cellunder conditions suitable for producing the recombinant protein.

In further aspects of the above methods, the host cell is selected fromthe group consisting of Pichia pastoris, Pichia finlandica, Pichiatrehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta(Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stipitis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Schizosacchromyces pombe,Schizosacchroyces sp. Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum,Physcomitrella patens and Neurospora crassa. Pichia sp., anySaccharomyces sp., any Schizosacchromyces sp., Hansenula polymorpha, anyKluyveromyces sp., Candida albicans, any Aspergillus sp., Trichodermareesei, Chrysosporium lucknowense, any Fusarium sp. and Neurosporacrassa.

Further provided are recombinant proteins produced by the host cellsdisclosed herein.

In particular embodiments, any one of the aforementioned host cells canfurther include genetic modifications that enable the host cells toproduce glycoproteins have predominantly particular N-glycan structuresthereon or particular mixtures of N-glycan structures thereon. Forexample, the host cells have been genetically engineered to produceN-glycans having a Man₃GlcNAc₂ or Man₅GlcNAc₂ core structure, which inparticular aspects include one or more additional sugars such as GlcNAc,Galactose, or sialic acid on the non-reducing end, and optionally fucoseon the GlcNAc at the reducing end. Thus, the N-glycans include bothbi-antennary and multi-antennary glycoforms and glycoforms that arebisected. Examples of N-glycans include but are not limited toMangGlcNAc₂, Man₇GlcNAc₂, Man₆GlcNAc₂, Man₅GlcNAc₂, GlcNAcMan₅GlcNAc₂,GalGlcNAcMan₅GlcNAc₂, NANAGaIGlcNAcMan₅GlcNAc₂, Man₃GlcNAc₂,GlcNAc₍₁₋₄₎Man₃GlcNAc₂, Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂,NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂.

DEFINITIONS

Unless otherwise defined herein, scientific and technical terms andphrases used in connection with the present invention shall have themeanings that are commonly understood by those of ordinary skill in theart. Further, unless otherwise required by context, singular terms shallinclude the plural and plural terms shall include the singular.Generally, nomenclatures used in connection with, and techniques ofbiochemistry, enzymology, molecular and cellular biology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer,Introduction to Glycobiology, Oxford Univ. Press (2003); WorthingtonEnzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbookof Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbookof Biochemistry: Section A Proteins, Vol II, CRC Press (1976);Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).

All publications, patents and other references mentioned herein arehereby incorporated by reference in their entireties.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the terms “N-glycan” and “glycoform” are usedinterchangeably and refer to an N-linked oligosaccharide, e.g., one thatis attached by an asparagine-N-acetylglucosamine linkage to anasparagine residue of a polypeptide. N-linked glycoproteins contain anN-acetylglucosamine residue linked to the amide nitrogen of anasparagine residue in the protein. The predominant sugars found onglycoproteins are glucose, galactose, mannose, fucose,N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialicacid (e.g., N-acetyl-neuraminic acid (NANA)). The processing of thesugar groups occurs cotranslationally in the lumen of the ER andcontinues in the Golgi apparatus for N-linked glycoproteins.

N-glycans have a common pentasaccharide core of Man₃GlcNAc₂ (“Man”refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). N-glycans differ withrespect to the number of branches (antennae) comprising peripheralsugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are addedto the Man₃GlcNAc₂ (“Man3”) core structure which is also referred to asthe “trimannose core”, the “pentasaccharide core” or the “paucimannosecore”. N-glycans are classified according to their branched constituents(e.g., high mannose, complex or hybrid). A “high mannose” type N-glycanhas five or more mannose residues. A “complex” type N-glycan typicallyhas at least one GlcNAc attached to the 1,3 mannose arm and at least oneGlcNAc attached to the 1,6 mannose arm of a “trimannose” core. ComplexN-glycans may also have galactose (“Gal”) or N-acetylgalactosamine(“GalNAc”) residues that are optionally modified with sialic acid orderivatives (e.g., “NANA” or “NeuAc”, where “Neu” refers to neuraminicacid and “Ac” refers to acetyl). Complex N-glycans may also haveintrachain substitutions comprising “bisecting” GlcNAc and core fucose(“Fuc”). Complex N-glycans may also have multiple antennae on the“trimannose core,” often referred to as “multiple antennary glycans.” A“hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3mannose arm of the trimannose core and zero or more mannoses on the 1,6mannose arm of the trimannose core. The various N-glycans are alsoreferred to as “glycoforms.”

Abbreviations used herein are of common usage in the art, see, e.g.,abbreviations of sugars, above. Other common abbreviations include“PNGase”, or “glycanase” or “glucosidase” which all refer to peptideN-glycosidase F (EC 3.2.2.18).

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid molecule to whichit has been linked. One type of vector is a “plasmid vector”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments may be ligated. Other vectors include cosmids, bacterialartificial chromosomes (BAC) and yeast artificial chromosomes (YAC).Another type of vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome (discussed in more detailbelow). Certain vectors are capable of autonomous replication in a hostcell into which they are introduced (e.g., vectors having an origin ofreplication which functions in the host cell). Other vectors can beintegrated into the genome of a host cell upon introduction into thehost cell, and are thereby replicated along with the host genome.Moreover, certain preferred vectors are capable of directing theexpression of genes to which they are operatively linked. Such vectorsare referred to herein as “recombinant expression vectors” (or simply,“expression vectors”).

As used herein, the term “sequence of interest” or “gene of interest”refers to a nucleic acid sequence, typically encoding a protein, that isnot normally produced in the host cell. The methods disclosed hereinallow efficient expression of one or more sequences of interest or genesof interest stably integrated into a host cell genome. Non-limitingexamples of sequences of interest include sequences encoding one or morepolypeptides having an enzymatic activity, e.g., an enzyme which affectsN-glycan synthesis in a host such as mannosyltransferases,N-acetylglucosaminyltransferases, UDP-N-acetylglucosamine transporters,galactosyltransferases, UDP-N-acetylgalactosyltransferase,sialyltransferases and fucosyltransferases.

The term “marker sequence” or “marker gene” refers to a nucleic acidsequence capable of expressing an activity that allows either positiveor negative selection for the presence or absence of the sequence withina host cell. For example, the Pichia pastoris URA5 gene is a marker genebecause its presence can be selected for by the ability of cellscontaining the gene to grow in the absence of uracil. Its presence canalso be selected against by the inability of cells containing the geneto grow in the presence of 5-FOA. Marker sequences or genes do notnecessarily need to display both positive and negative selectability.Non-limiting examples of marker sequences or genes from Pichia pastorisinclude ADE1, ARG4, HIS4 and URA3. For antibiotic resistance markergenes, kanamycin, neomycin, geneticin (or G418), paromomycin andhygromycin resistance genes are commonly used to allow for growth in thepresence of these antibiotics.

“Operatively linked” expression control sequences refers to a linkage inwhich the expression control sequence is contiguous with the gene ofinterest to control the gene of interest, as well as expression controlsequences that act in trans or at a distance to control the gene ofinterest.

The term “expression control sequence” or “regulatory sequences” areused interchangeably and as used herein refer to polynucleotidesequences which are necessary to affect the expression of codingsequences to which they are operatively linked. Expression controlsequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “recombinant host cell” (“expression host cell”, “expressionhost system”, “expression system” or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinant vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

The term “eukaryotic” refers to a nucleated cell or organism, andincludes insect cells, plant cells, mammalian cells, animal cells andlower eukaryotic cells.

The term “lower eukaryotic cells” includes yeast and filamentous fungi.Yeast and filamentous fungi include, but are not limited to: Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae,Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichialindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria,Pichia guercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica,Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Schizosacchromyces pombe, Schizosacchroyces sp., Hansenula polymorpha,Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei,Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusariumvenenatum, Physcomitrella patens and Neurospora crassa. Pichia sp., anySaccharomyces sp., any Schizosacchromyces sp., Hansenula polymorpha, anyKluyveromyces sp., Candida albicans, any Aspergillus sp., Trichodermareesei, Chrysosporium lucknowense, any Fusarium sp. and Neurosporacrassa.

The function of a gene encoding a protein is said to be ‘reduced’ whenthat gene has been modified, for example, by deletion, insertion,mutation or substitution of one or more nucleotides, such that themodified gene encodes a protein which has at least 20% to 50% loweractivity, in particular aspects, at least 40% lower activity or at least50% lower activity, when measured in a standard assay, as compared tothe protein encoded by the corresponding gene without such modification.The function of a gene encoding a protein is said to be ‘eliminated’when the gene has been modified, for example, by deletion, insertion,mutation or substitution of one or more nucleotides, such that themodified gene encodes a protein which has at least 90% to 99% loweractivity, in particular aspects, at least 95% lower activity or at least99% lower activity, when measured in a standard assay, as compared tothe protein encoded by the corresponding gene without such modification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.All publications and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting in any manner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates representative results from deep-well plate screeningwhere human anti-DKK1 antibody is produced in Pichia pastoris host cellsin which the endogenous PDI1 gene is expressed (Panel A), both in thepresence of the endogenous PDI1 gene and the human PDI gene (Panel B),and in a cell line expressing the human PDI gene and in which theendogenous PDI1 gene function has been knocked out (Panel C).

FIG. 2 illustrates the action of human PDI and its co-chaperones inthiol-redox reactions in the endoplasmic reticulum.

FIGS. 3A and 3B show the genealogy of yeast strains described in theexamples for illustrating the invention.

FIGS. 4A and 48 shows representative results from shakeflask (A) and 0.5L bioreactor (B) expression studies in which human anti-Her2 antibodywas produced in Pichia pastoris strains in which the human PDI gene(hPDI) replaced the endogenous PDI1 and strains in which the human PDIreplaced the endogenous PDI1 and the PMT1 gene is disrupted(hPDI+Δpmt1). Antibodies were recovered and resolved by polyacrylamidegel electrophoresis on non-reducing and reducing polyacrylamide gels.Lanes 1-2 shows antibodies produced from two clones produced fromtransformation of strain yGLY2696 with plasmid vector pGLY2988 encodingthe anti-Her2 antibody and lanes 3-6 shows the antibodies produced fromfour clones produced from transformation of strain yGLY2696 in which thePMT1 gene was deleted and with plasmid vector pGLY2988 encoding theanti-Her2 antibody.

FIG. 5 shows representative results from a shakeflask expression studyin which human anti-DKK1 antibody was produced in Pichia pastorisstrains in which the human PDI (hPDI) gene replaced the endogenous PDI1and strains in which the human PDI replaced the endogenous PDI1 and thePMT1 gene disrupted (hPDI+Δpmt1). Antibodies were recovered and resolvedby polyacrylamide gel electrophoresis on non-reducing and reducingpolyacrylamide gels. Lanes 1 and 3 shows antibodies produced from twoclones produced from transformation of strains yGLY2696 and yGLY2690with plasmid vector pGLY2260 encoding the anti-DKK1 antibody and lanes 2and 4 shows the antibodies produced from two clones produced fromtransformation of strains yGLY2696 and yGLY2690 in which the PMT1 genewas deleted with plasmid vector pGLY2260 encoding the anti-DKK1antibody.

FIG. 6 shows results from a 0.5 L bioreactor expression study wherehuman anti-Her2 antibody is produced in Pichia pastoris strains in whichthe human PDI gene (hPDI) replaced the endogenous PDI1, strains in whichthe human PDI replaced the endogenous PDI1 and the PMT4 gene disrupted(hPDI+Δpmt4), and strains that express only the endogenous PDI1 but inwhich the PMT4 gene is disrupted (PpPDI+Δpmt4). Antibodies wererecovered and resolved by polyacrylamide gel electrophoresis onnon-reducing polyacrylamide gels. Lanes 1 and 2 shows antibodiesproduced from two clones from transformation of strain yGLY24-1 withplasmid vector pGLY2988 encoding the anti-Her2 antibody and lanes 3-5show anti-Her2 antibodies produced from three clones produced fromtransformation of strain yGLY2690 in which the PMT4 gene was deleted.

FIG. 7 shows results from a shakeflask expression study where humananti-CD20 antibody is produced in Pichia pastoris strains in which thehuman PDI replaced the endogenous PDI1 and the PMT4 gene is disrupted(hPDI+Δpmt4) and strains that express only the endogenous PDI1 but inwhich the PMT4 gene is disrupted (PpPDI+Δpmt4). Antibodies wererecovered and resolved by polyacrylamide gel electrophoresis onnon-reducing and reducing polyacrylamide gels Lane 1 shows antibodiesproduced from strain yGLY24-1 transformed with plasmid vector pGLY3200encoding the anti-CD20 antibody; lanes 2-7 show anti-CD20 antibodiesproduced from six clones produced from transformation of strain yGLY2690in which the PMT4 gene was deleted.

FIG. 8 illustrates the construction of plasmid vector pGLY642 encodingthe human PDI (hPDI) and targeting the Pichia pastoris PDI1 locus.

FIG. 9 illustrates the construction of plasmid vector pGLY2232 encodingthe human ERO1α (hERO1α) and targeting the Pichia pastoris PrB1 locus.

FIG. 10 illustrates the construction of plasmid vector pGLY2233 encodingthe human GRP94 and targeting the Pichia pastoris PEP4 locus.

FIG. 11 illustrates the construction of plasmid vector pGFI207t encodingthe T. reesei α-1,2 mannosidase (TrMNS1) and mouse α-1,2 mannosidase IA(FB53) and targeting the Pichia pastoris PRO locus.

FIG. 12 illustrates the construction of plasmid vector pGLY1162 encodingthe T. reesei α-1,2 mannosidase (TrMNS1) and targeting the Pichiapastoris PRO locus.

FIG. 13 is maps of plasmid vector pGLY2260 and 2261 encoding theanti-DKK1 antibody heavy chain (GFI710H) and light chain (GFI710L) ortwo light chains (GFI710L) and targeting the Pichia pastoris TRP2 locus.

FIG. 14 is a map of plasmid vector pGLY2012 encoding the anti-ADDLantibody heavy chain (Hc) and light chain (Lc) and targeting the Pichiapastoris TRP2 locus.

FIG. 15 is a map of plasmid vector pGLY2988 encoding the anti-HER2antibody (anti-HER2) heavy chain (Hc) and light chain (Lc) and targetingthe Pichia pastoris TRP2 locus.

FIG. 16 is a map of plasmid vector pGLY3200 encoding the anti-CD20antibody heavy chain (Hc) and light chain (Lc) and targeting the Pichiapastoris TRP2 locus.

FIG. 17 is a map of plasmid vector pGLY3822 encoding the Pichia pastorisPMR1 and targeting the Pichia pastoris URA6 locus.

FIG. 18 is a map of plasmid vector pGLY3827 encoding the Arabidopsisthaliana ECA1 (AtECA1) and targeting the Pichia pastoris URA6 locus.

FIG. 19 is a map of plasmid vector pGLY1234 encoding the human CRT(hCRT) and human ERp57(hERp57) and targeting the Pichia pastoris HIS3locus.

DETAILED DESCRIPTION OF THE INVENTION

Molecular chaperones play a critical role in the folding and secretionof antibodies. One chaperone protein in particular, Protein DisulfideIsomerase (PDI), functions to catalyze inter and intra disulphide bondformation that link the antibody heavy and light chains. Proteindisulfide isomerase (PDI) can produce a substantial increase or asubstantial decrease in the recovery of disulfide-containing proteins,when compared with the uncatalyzed reaction; a high concentration of PDIin the endoplasmic reticulum (ER) is essential for the expression ofdisulfide-containing proteins [Puig and Gilbert, J. Biol. Chem.,269:7764-7771 (1994)]. Past attempts to increase antibody expressionlevels in Pichia pastoris by overexpressing human PDI chaperone proteinand/or overexpressing endogenous PDI1 have been with limited success. Wehave undertaken humanization of the chaperone pathway in Pichia pastoristo explore the possibility of antibody yield improvement through directgenetic engineering.

We have found in a Pichia pastoris model that replacement of the yeastgene encoding the endogenous PDI1 protein with an expression cassetteencoding a heterologous PDI protein resulted in approximately afive-fold improvement in the yield of recombinant human antibodyproduced by the recombinant yeast cells as compared to the yieldproduced by recombinant yeast cells that expressed only the endogenousPDI1 protein and about a three-fold increase in yield compared to theyield produced by recombinant yeast cells that co-expressed theheterologous PDI protein with the endogenous PDI1 protein.

Without being limited to any scientific theory of the mechanism of theinvention, it is believed that heterologous recombinant proteins mayinteract more efficiently with heterologous chaperone proteins than hostcell chaperone proteins in the course of their folding and assemblyalong the secretory pathway. In the case of co-expression, theheterologous chaperone protein may compete with the endogenous chaperoneprotein for its substrate, i.e., heterologous recombinant proteins. Itis further believed that the heterologous PDI protein and recombinantprotein be from the same species. Therefore, replacement of the geneencoding the endogenous chaperone protein with an expression cassetteencoding a heterologous chaperone may be a better means for producingrecombinant host cells for producing recombinant proteins that merelyco-expressing the heterologous chaperone protein with the endogenouschaperone protein.

In addition, further improvements in recombinant protein yield may beobtained by overexpressing in the recombinant host cell the heterologousPDI protein and an additional heterologous co-chaperone proteins, suchas ERO1α and or the GRP94 proteins. In further aspects, the recombinanthost cell can further overexpress FAD, FLC1, and ERp44 proteins. Sincethese genes are related in function, it may be desirable to include thenucleic acid molecules that encode these genes in a single vector, whichtransformed into the host cell. Expression of the proteins may beeffected by operably linking the nucleic acid molecules encoding theproteins to a heterologous or homologous promoter. In particularaspects, when the host cell is Pichia pastoris, expression of one ormore of the heterologous co-chaperone proteins may be effected by ahomologous promoter such as the KAR2 promoter or a promoter from anotherER-specific gene. In further aspects, all of the heterologous chaperoneproteins and recombinant protein be from the same species.

As exemplified in the Examples using Pichia pastoris as a model, themethods disclosed herein are particularly useful in the production ofrecombinant human glycoproteins, including antibodies, from lowereukaryotic host cells, such as yeast and filamentous fungi. For example,secretion of recombinant proteins from Pichia pastoris proceeds moreefficiently as the folding and assembly of the protein of interest isassisted by human PDI, and optionally including other mammalian-derivedchaperone proteins, such as ERO1α and GRP94, thereby improving yield. Asexemplified in the Examples, the methods herein will especially benefitantibody production in which the heavy and light chains must be properlyassembled through disulphide bonds in order to achieve activity.

Thus, there methods herein provide significant advantages with respectto addressing the problem of low productivity in the secretion ofrecombinant antibodies from lower eukaryotic host cells, and inparticular yeast and filamentous fungi, for example, Pichia pastoris. Inthe past, yeast, human or mouse chaperone proteins were overexpressedwith limited success while the present invention demonstrates thatimproved productivity of correctly folded and secreted heterologousproteins, such as antibodies, can be obtained through replacement of thehost cells' endogenous chaperone proteins with heterologous chaperoneproteins. The overexpression of mammalian-derived chaperone proteins,combined with the deletion of the endogenous gene encoding a proteinhomolog unexpectedly results in improved productivity of glycoproteins,compared with overexpression of the mammalian-derived protein alone.

We further found that host cells, transformed with nucleic acidmolecules encoding one or more chaperone genes as described above, canbe further genetically manipulated to improve other characteristics ofthe recombinant proteins produced therefrom. This is especially true inthe case of recombinant mammalian glycoprotein production from lowereukaryotic host cells such as yeast or filamentous fungi.

For example, lower eukaryotic cells such as Saccharomyces cerevisiae,Candida albicans, and Pichia pastoris, contain a family of genes knownas protein O-mannosyltransferases (PMTs) involved in the transfer ofmannose to seryl and threonyl residues of secretory proteins. We foundthat Pichia pastoris cell lines, which have been genetically altered toexpress one or more humanized or chimeric chaperone genes, are betterable to tolerate deletion of one or more PMT genes, with little or noeffect on cell growth or protein expression. PMT genes which may bedeleted include PMT1, PMT2, PMT4, PMT5, and PMT6. In general, Pichiapastoris host cells in which both the OCH1 gene and the PMT gene isdeleted either grow poorly or not at all. Deletion or functionalknockout of the OCH1 gene is necessary for constructing recombinantPichia pastoris host cells that can make human glycoproteins that havehuman-like N-glycans. Because it is desirable to produce humanglycoproteins that have no or reduced O-glycosylation, there has been aneed to find means for reducing O-glycosylation in recombinant Pichiapastoris host cells that are also capable of producing humanglycoproteins with human-like N-glycans. We found that Pichia pastorishost cells containing one or more chaperone genes as disclosed hereincan be further genetically altered to contain a deletion or functionalknockout of the OCH1 gene and a deletion or functional knockout of oneor more PMT genes, such as PMT1, PMT4, PMT5, and/or PMT6. Theserecombinant cells are viable and produce human glycoproteins withhuman-like N-glycans in high yield and with reduced O-glycosylation. Inaddition, a further reduction in O-glycosylation was achieved by growingthe cells in the presence of a PMT protein inhibitor.

As exemplified in the Examples, we demonstrate that the methodsdisclosed herein are particularly useful in the production ofrecombinant human glycoproteins, including antibodies, from lowereukaryotic host cells, such as yeast and filamentous fungi with improvedproperties, since the host cells of the present invention exhibittolerance to chemical PMT protein inhibitors and/or deletion of PMTgenes. The Examples show that the recombinant proteins have reducedO-glycosylation occupancy and length of O-glycans compared with priorlower eukaryotic expression systems. As exemplified in the Examples, themethods herein will especially benefit antibody production in which theheavy and light chains must be properly assembled through disulphidebonds in order to achieve activity and the antibodies must have reducedor no O-glycosylation.

We have further found that over-expression of Pichia pastoris Golgi Ca²⁺ATPase (PpPMR1) or Arabidopsis thaliana ER Ca²⁺ ATPase (AtECA1) effectedabout a 2-fold reduction in O-glycan occupancy compared to the abovestrains wherein the endogenous PDI1 had been replaced with the human PDIbut which did not express either Ca²⁺ ATPase. Thus, in furtherembodiments, any one of the host cells disclosed herein can furtherinclude one or more nucleic acid molecules encoding an endogenous orexogenous Golgi or ER Ca²⁺ ATPase, wherein the Ca²⁺ ATPase is operablylinked to a heterologous promoter. These host cells can be used toproduce glycoproteins with reduced O-glycosylation.

Calreticulin (CRT) is a multifunctional protein that acts as a majorCa(2+)-binding (storage) protein in the lumen of the endoplasmicreticulum. It is also found in the nucleus, suggesting that it may havea role in transcription regulation. Calreticulin binds to the syntheticpeptide KLGFFKR. (SEQ ID NO:75), which is almost identical to an aminoacid sequence in the DNA-binding domain of the superfamily of nuclearreceptors. Calreticulin binds to antibodies in certain sera of systemiclupus and Sjogren patients which contain anti-Ro/SSA antibodies, it ishighly conserved among species, and it is located in the endoplasmic andsarcoplasmic reticulum where it may bind calcium. Calreticulin binds tomisfolded proteins and prevents them from being exported from theEndoplasmic reticulum to the Golgi apparatus.

ERp57 is a chaperone protein of the endoplasmic reticulum that interactswith lectin chaperones calreticulin and calnexin to modulate folding ofnewly synthesized glycoproteins. The protein was once thought to be aphospholipase; however, it has been demonstrated that the proteinactually has protein disulfide isomerase activity. Thus, the ERp57 is alumenal protein of the endoplasmic reticulum (ER) and a member of theprotein disulfide isomerase (PDI) family. It is thought that complexesof lectins and this protein mediate protein folding by promotingformation of disulfide bonds in their glycoprotein substrates. Incontrast to archetypal PDI, ERp57 interacts specifically with newlysynthesized glycoproteins.

We have further found that over-expression of the human CRT and humanERp57 in Pichia pastoris effected about a one-third reduction inO-glycan occupancy compared to strains wherein the endogenous PDI1 hadbeen replaced with the human PDI but which did not express the hCRT andhERp57. Thus, in further embodiments, any one of the host cells hereincan further include one or more nucleic acid molecules encoding acalreticulin and an ERp57 protein, each operably linked to aheterologous promoter. These host cells can be used to produceglycoproteins with reduced O-glycosylation.

Thus, the methods herein provide significant advantages with respect toaddressing the problem of low productivity in the secretion ofrecombinant antibodies from lower eukaryotic host cells, and inparticular yeast and filamentous fungi, for example, Pichia pastoris. Inthe past, yeast, human or mouse chaperone proteins were overexpressedwith limited success while the present invention demonstrates thatimproved productivity of correctly folded and secreted heterologousproteins, such as antibodies, can be obtained through replacement of thehost cells' endogenous chaperone proteins with heterologous chaperoneproteins. The overexpression of mammalian-derived chaperone proteins,combined with the deletion of the endogenous gene encoding a proteinhomolog unexpectedly results in improved productivity of glycoproteins,compared with overexpression of the mammalian-derived protein alone.

Therefore, the present invention provides methods for increasingproduction of an overexpressed gene product present in a lower eukaryotehost cell, which includes expressing a heterologous chaperone protein inthe host cell in place of an endogenous chaperone protein and therebyincreasing production of the overexpressed gene product. Also providedis a method of increasing production of an overexpressed gene productfrom a host cell by disrupting or deleting a gene encoding an endogenouschaperone protein and expressing a nucleic acid molecule encoding aheterologous chaperone protein encoded in an expression vector presentin or provided to the host cell, thereby increasing the production ofthe overexpressed gene product. Further provided is a method forincreasing production of overexpressed gene products from a host cell,which comprises expressing at least one heterologous chaperone proteinin the host cell in place of the endogenous chaperone protein. In thepresent context, an overexpressed gene product is one which is expressedat levels greater than normal endogenous expression for that geneproduct.

In one embodiment, the method comprises deleting or disruptingexpression of an endogenous chaperone protein and effecting theexpression of one or more heterologous chaperone proteins and anoverexpressed gene product in a host cell, and cultivating said hostcell under conditions suitable for secretion of the overexpressed geneproduct. The expression of the chaperone protein and the overexpressedgene product can be effected by inducing expression of a nucleic acidmolecule encoding the chaperone protein and a nucleic acid moleculeencoding the overexpressed gene product wherein said nucleic acidmolecules are present in a host cell.

In another embodiment, the expression of the heterologous chaperoneprotein and the overexpressed gene product are effected by introducing afirst nucleic acid molecule encoding a heterologous chaperone proteinand a second nucleic acid molecule encoding a gene product to beoverexpressed into a host cell in which expression of at least one geneencoding an endogenous chaperone protein has been disrupted or deletedunder conditions suitable for expression of the first and second nucleicacid molecules. In further aspects, one or both of said first and secondnucleic acid molecules are present in expression vectors. In furtheraspects, one or both of said first and second nucleic acid molecules arepresent in expression/integration vectors. In a further embodiment,expression of the heterologous chaperone protein is effected by inducingexpression of the nucleic acid molecule encoding the chaperone proteinwherein the nucleic acid molecule into a host cell in which the geneencoding the endogenous chaperone protein has been deleted or disrupted.Expression of the second protein is effected by inducing expression of anucleic acid molecule encoding the gene product to be overexpressed byintroducing a nucleic acid molecule encoding said second gene productinto the host cell.

The present invention further provides methods for increasing productionof an overexpressed gene product present in a lower eukaryote host cellwith reduced O-glycosylation, which includes expressing a heterologouschaperone protein in the host cell in place of an endogenous chaperoneprotein and wherein the host cell has had one or more genes in theprotein O-mannosyltransferase (PMT) family disrupted or deleted, therebyincreasing production of the overexpressed gene product with reducedO-glycosylation. Also provided is a method of increasing production ofan overexpressed gene product with reduced O-glycosylation from a hostcell by disrupting or deleting a gene encoding an endogenous chaperoneprotein and a gene encoding a PMT and expressing a nucleic acid moleculeencoding a heterologous chaperone protein encoded in an expressionvector present in or provided to the host cell, thereby increasing theproduction of the overexpressed gene product. Further provided is amethod for increasing production of overexpressed gene products withreduced O-glycosylation from a host cell, which comprises expressing atleast one heterologous chaperone protein in the host cell in place ofthe endogenous chaperone protein and wherein at least one PMT gene hasbeen disrupted or deleted.

In one embodiment, the method comprises deleting or disruptingexpression of at least one endogenous chaperone protein and at least onePMT gene and effecting the expression of one or more heterologouschaperone proteins and an overexpressed gene product in a host cell, andcultivating said host cell under conditions suitable for secretion ofthe overexpressed gene product with reduced O-glycosylation. Theexpression of the chaperone protein and the overexpressed gene productcan be effected by inducing expression of a nucleic acid moleculeencoding the chaperone protein and a nucleic acid molecule encoding theoverexpressed gene product wherein said nucleic acid molecules arepresent in a host cell.

In another embodiment, the expression of the heterologous chaperoneprotein and the overexpressed gene product are effected by introducing afirst nucleic acid molecule encoding a heterologous chaperone proteinand a second nucleic acid molecule encoding a gene product to beoverexpressed into a host cell in which expression of at least one geneencoding an endogenous chaperone protein and at least one PMT gene havebeen disrupted or deleted under conditions suitable for expression ofthe first and second nucleic acid molecules. In further aspects, one orboth of said first and second nucleic acid molecules are present inexpression vectors. In further aspects, one or both of said first andsecond nucleic acid molecules are present in expression/integrationvectors. In a further embodiment, expression of the heterologouschaperone protein is effected by inducing expression of the nucleic acidmolecule encoding the chaperone protein wherein the nucleic acidmolecule into a host cell in which the gene encoding the endogenouschaperone protein has been deleted or disrupted. Expression of thesecond protein is effected by inducing expression of a nucleic acidmolecule encoding the gene product to be overexpressed by introducing anucleic acid molecule encoding said second gene product into the hostcell.

In a further aspect of any one of the above embodiments, theheterologous chaperone protein corresponds in species or class to theendogenous chaperone protein. For example, if the host cell is a yeastcell and the endogenous chaperone protein is a protein disulfideisomerase (PDI) then the corresponding heterologous PDI can be amammalian PDI. In further still aspects of any one of the aboveembodiments, the heterologous chaperone proteins expressed in aparticular host cell are from the same species as the species for theoverexpressed gene product. For example, if the overexpressed geneproduct is a human protein then the heterologous chaperone proteins arehuman chaperone proteins; or if the overexpressed gene product is abovine protein then the heterologous chaperone protein is a bovinechaperone protein.

Chaperone proteins include any chaperone protein which can facilitate orincrease the secretion of proteins. In particular, members of theprotein disulfide isomerase and heat shock 70 (hsp70) families ofproteins are contemplated. An uncapitalized “hsp70” is used herein todesignate the heat shock protein 70 family of proteins which sharestructural and functional similarity and whose expression are generallyinduced by stress. To distinguish the hsp70 family of proteins from thesingle heat shock protein of a species which has a molecular weight ofabout 70,000, and which has an art-recognized name of heat shockprotein-70, a capitalized HSP70 is used herein. Accordingly, each memberof the hsp70 family of proteins from a given species has structuralsimilarity to the HSP70 protein from that species.

The present invention is directed to any chaperone protein having thecapability to stimulate secretion of an overexpressed gene product. Themembers of the hsp70 family of proteins are known to be structurallyhomologous and include yeast hsp70 proteins such as KAR2, HSP70, BiP,SSA1-4, SSBI, SSC1 and SSD1 gene products and eukaryotic hsp70 proteinssuch as HSP68, HSP72, HSP73, HSC70, clathrin uncoating ATPase, IgG heavychain binding protein (BiP), glucose-regulated proteins 75, 78 and 80(GRP75, GRP78 and GRP80) and the like. Moreover, according to thepresent invention any hsp70 chaperone protein having sufficient homologyto the yeast KAR2 or mammalian BiP polypeptide sequence can be used inthe present methods to stimulate secretion of an overexpressed geneproduct. Members of the PDI family are also structurally homologous, andany PDI which can be used according to the present method iscontemplated herein. In particular, mammalian (including human) andyeast PDI, prolyl-4-hydroxylase β-subunit, ERp57, ERp29, ERp72, GSBP,ERO1α, GRP94, GRP170, BiP, and T3BP and yeast EUG1 are contemplated.Because many therapeutic proteins for use in human are of human origin,a particular aspect of the methods herein is that the heterologouschaperone protein is of human origin. In further still embodiments, thepreferred heterologous chaperone protein is a PDI protein, particularlya PDI protein of human origin.

Attempts to increase expression levels of heterologous human proteins inyeast cell lines by overexpressing human BiP, using constitutivepromoters such as GAPDH, have been largely unsuccessful. Knockouts ofPichia pastoris KAR2, the homolog of human BiP, have been harmful tocells. The limitations of the prior art can be overcome by constructinga chimeric BiP gene, in which the human ATPase domain is replaced by theATPase domain of Pichia pastoris KAR2, fused to the human BiP peptidebinding domain, under the control of the KAR2, or other ER-specificpromoter from Pichia pastoris. Further improvements in yield may beobtained by combining the replacement of the endogenous PDI1 gene, asdescribed above, with the use of chimeric BiP and human ERdj3.

In further aspects, the overexpressed gene product is a secreted geneproduct. Procedures for observing whether an overexpressed gene productis secreted are readily available to the skilled artisan. For example,Goeddel, (Ed.) 1990, Gene Expression Technology, Methods in Enzymology,Vol 185, Academic Press, and Sambrook et al. 1989, Molecular Cloning: ALaboratory Manual, Vols. 1-3, Cold Spring Harbor Press, N.Y., provideprocedures for detecting secreted gene products.

To secrete an overexpressed gene product the host cell is cultivatedunder conditions sufficient for secretion of the overexpressed geneproduct. Such conditions include temperature, nutrient and cell densityconditions that permit secretion by the cell. Moreover, such conditionsare conditions under which the cell can perform basic cellular functionsof transcription, translation and passage of proteins from one cellularcompartment to another and are known to the skilled artisan.

Moreover, as is known to the skilled artisan a secreted gene product canbe detected in the culture medium used to maintain or grow the presenthost cells. The culture medium can be separated from the host cells byknown procedures, for example, centrifugation or filtration. Theoverexpressed gene product can then be detected in the cell-free culturemedium by taking advantage of known properties characteristic of theoverexpressed gene product. Such properties can include the distinctimmunological, enzymatic or physical properties of the overexpressedgene product. For example, if an overexpressed gene product has a uniqueenzyme activity an assay for that activity can be performed on theculture medium used by the host cells. Moreover, when antibodiesreactive against a given overexpressed gene product are available, suchantibodies can be used to detect the gene product in any knownimmunological assay (See Harlowe, et al., 1988, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press)

In addition, a secreted gene product can be a fusion protein wherein thegene product includes a heterologous signal or leader peptide thatfacilitates the secretion of the gene product. Secretion signal peptidesare discrete amino acid sequences, which cause the host cell to direct agene product through internal and external cellular membranes and intothe extracellular environment. Secretion signal peptides are present atthe N-terminus of a nascent polypeptide gene product targeted forsecretion. Additional eukaryotic secretion signals can also be presentalong the polypeptide chain of the gene product in the form ofcarbohydrates attached to specific amino acids, i.e. glycosylationsecretion signals.

N-terminal signal peptides include a hydrophobic domain of about 10 toabout 30 amino acids which can be preceded by a short charged domain ofabout two to about 10 amino acids. Moreover, the signal peptide ispresent at the N-terminus of gene products destined for secretion. Ingeneral, the particular sequence of a signal sequence is not criticalbut signal sequences are rich in hydrophobic amino acids such as alanine(Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro),phenylalanine (Phe), tryptophan (Trp), methionine (Met) and the like.

Many signal peptides are known (Michaelis et al., Ann. Rev. Microbiol.36: 425 (1982). For example, the yeast acid phosphatase, yeastinvertase, and the yeast α-factor signal peptides have been attached toheterologous polypeptide coding regions and used successfully forsecretion of the heterologous polypeptide (See for example, Sato et al.Gene 83: 355-365 (1989); Chang et al. Mol. Cell. Biol. 6: 1812-1819(1986); and Brake et al. Proc. Natl. Acad. Sci. USA 81: 4642-4646(1984). Therefore, the skilled artisan can readily design or obtain anucleic acid molecule which encodes a coding region for an overexpressedgene product which also has a signal peptide at the 5′-end.

Examples of overexpressed gene products which are preferably secreted bythe present methods include mammalian gene products such as enzymes,cytokines, growth factors, hormones, vaccines, antibodies and the like.More particularly, overexpressed gene products include but are notlimited to gene products such as erythropoietin, insulin, somatotropin,growth hormone releasing factor, platelet derived growth factor,epidermal growth factor, transforming growth factor α, transforminggrowth factor β, epidermal growth factor, fibroblast growth factor,nerve growth factor, insulin-like growth factor I, insulin-like growthfactor II, clotting Factor VIII, superoxide dismutase, α-interferon,γ-interferon, interleukin-1, interleukin-2, interleukin-3,interleukin-4, interleukin-5, interleukin-6, granulocyte colonystimulating factor, multi-lineage colony stimulating activity,granulocyte-macrophage stimulating factor, macrophage colony stimulatingfactor, T cell growth factor, lymphotoxin, immunoglobulins, antibodies,and the like. Further included are fusion proteins, including but notlimited to, peptides and polypeptides fused to the constant region of animmunoglobulin or antibody. Particularly useful overexpressed geneproducts are human gene products.

The terms “antibody”, “antibodies”, and “immunoglobulin(s)” encompassany recombinant monoclonal antibody produced by recombinant DNAtechnology and further is meant to include humanized and chimericantibodies.

The present methods can readily be adapted to enhance secretion of anyoverexpressed gene product which can be used as a vaccine. Overexpressedgene products which can be used as vaccines include any structural,membrane-associated, membrane-bound or secreted gene product of amammalian pathogen. Mammalian pathogens include viruses, bacteria,single-celled or multi-celled parasites which can infect or attack amammal. For example, viral vaccines can include vaccines against virusessuch as human immunodeficiency virus (HIV), R. rickettsii, vaccinia,Shigella, poliovirus, adenovirus, influenza, hepatitis A, hepatitis B,dengue virus, Japanese B encephalitis, Varicella zoster,cytomegalovirus, hepatitis A, rotavirus, as well as vaccines againstviral diseases like Lyme disease, measles, yellow fever, mumps, rabies,herpes, influenza, parainfluenza and the like. Bacterial vaccines caninclude vaccines against bacteria such as Vibrio cholerae, Salmonellatyphi, Bordetella pertussis, Streptococcus pneumoniae, Hemophilusinfluenza, Clostridium tetani, Corynebacterium diphtheriae,Mycobacterium leprae, Neisseria gonorrhoeae, Neisseria meningitidis,Coccidioides immitis, and the like.

In general, the overexpressed gene products and the heterologouschaperone proteins of the present invention are expressed recombinantly,that is, by placing a nucleic acid molecule encoding a gene product or achaperone protein into an expression vector. Such an expression vectorminimally contains a sequence which effects expression of the geneproduct or the heterologous chaperone protein when the sequence isoperably linked to a nucleic acid molecule encoding the gene product orthe chaperone protein. Such an expression vector can also containadditional elements like origins of replication, selectable markers,transcription or termination signals, centromeres, autonomousreplication sequences, and the like.

According to the present invention, first and second nucleic acidmolecules encoding an overexpressed gene product and a heterologouschaperone protein, respectively, can be placed within expression vectorsto permit regulated expression of the overexpressed gene product and/orthe heterologous chaperone protein. While the heterologous chaperoneprotein and the overexpressed gene product can be encoded in the sameexpression vector, the heterologous chaperone protein is preferablyencoded in an expression vector which is separate from the vectorencoding the overexpressed gene product. Placement of nucleic acidmolecules encoding the heterologous chaperone protein and theoverexpressed gene product in separate expression vectors can increasethe amount of secreted overexpressed gene product.

As used herein, an expression vector can be a replicable or anon-replicable expression vector. A replicable expression vector canreplicate either independently of host cell chromosomal DNA or becausesuch a vector has integrated into host cell chromosomal DNA. Uponintegration into host cell chromosomal DNA such an expression vector canlose some structural elements but retains the nucleic acid moleculeencoding the gene product or the chaperone protein and a segment whichcan effect expression of the gene product or the heterologous chaperoneprotein. Therefore, the expression vectors of the present invention canbe chromosomally integrating or chromosomally nonintegrating expressionvectors.

In a further embodiment, one or more heterologous chaperone proteins areoverexpressed in a host cell by introduction of integrating ornonintegrating expression vectors into the host cell. Followingintroduction of at least one expression vector encoding at least onechaperone protein, the gene product is then overexpressed by inducingexpression of an endogenous gene encoding the gene product, or byintroducing into the host cell an expression vector encoding the geneproduct. In another embodiment, cell lines are established whichconstitutively or inducibly express at least one heterologous chaperoneprotein. An expression vector encoding the gene product to beoverexpressed is introduced into such cell lines to achieve increasedsecretion of the overexpressed gene product.

The present expression vectors can be replicable in one host cell type,e.g., Escherichia coli, and undergo little or no replication in anotherhost cell type, e.g., a eukaryotic host cell, so long as an expressionvector permits expression of the heterologous chaperone proteins oroverexpressed gene products and thereby facilitates secretion of suchgene products in a selected host cell type.

Expression vectors as described herein include DNA or RNA moleculesengineered for controlled expression of a desired gene, that is, a geneencoding the present chaperone proteins or a overexpressed gene product.Such vectors also encode nucleic acid molecule segments which areoperably linked to nucleic acid molecules encoding the present chaperonepolypeptides or the present overexpressed gene products. Operably linkedin this context means that such segments can effect expression ofnucleic acid molecules encoding chaperone protein or overexpressed geneproducts. These nucleic acid sequences include promoters, enhancers,upstream control elements, transcription factors or repressor bindingsites, termination signals and other elements which can control geneexpression in the contemplated host cell. Preferably the vectors arevectors, bacteriophages, cosmids, or viruses.

Expression vectors of the present invention function in yeast ormammalian cells. Yeast vectors can include the yeast 2μ circle andderivatives thereof, yeast vectors encoding yeast autonomous replicationsequences, yeast minichromosomes, any yeast integrating vector and thelike. A comprehensive listing of many types of yeast vectors is providedin Parent et al. (Yeast 1: 83-138 (1985)).

Elements or nucleic acid sequences capable of effecting expression of agene product include promoters, enhancer elements, upstream activatingsequences, transcription termination signals and polyadenylation sites.All such promoter and transcriptional regulatory elements, singly or incombination, are contemplated for use in the present expression vectors.Moreover, genetically-engineered and mutated regulatory sequences arealso contemplated herein.

Promoters are DNA sequence elements for controlling gene expression. Inparticular, promoters specify transcription initiation sites and caninclude a TATA box and upstream promoter elements. The promotersselected are those which would be expected to be operable in theparticular host system selected. For example, yeast promoters are usedin the present expression vectors when a yeast host cell such asSaccharomyces cerevisiae, Kluyveromyces lactis, or Pichia pastoris isused whereas fungal promoters would be used in host cells such asAspergillus niger, Neurospora crassa, or Tricoderma reesei. Examples ofyeast promoters include but are not limited to the GAPDH, AOX1, GAL1,PGK, GAP, TPI, CYC1, ADH2, PHO5, CUP1, MFα1, PMA1, PDI, TEF, and GUT1promoters. Romanos et al. (Yeast 8: 423-488 (1992)) provide a review ofyeast promoters and expression vectors.

The promoters that are operably linked to the nucleic acid moleculesdisclosed herein can be constitutive promoters or inducible promoters.Inducible promoters, that is promoters which direct transcription at anincreased or decreased rate upon binding of a transcription factor.Transcription factors as used herein include any factor that can bind toa regulatory or control region of a promoter an thereby affecttranscription. The synthesis or the promoter binding ability of atranscription factor within the host cell can be controlled by exposingthe host to an inducer or removing an inducer from the host cell medium.Accordingly to regulate expression of an inducible promoter, an induceris added or removed from the growth medium of the host cell. Suchinducers can include sugars, phosphate, alcohol, metal ions, hormones,heat, cold and the like. For example, commonly used inducers in yeastare glucose, galactose, and the like.

Transcription termination sequences that are selected are those that areoperable in the particular host cell selected. For example, yeasttranscription termination sequences are used in the present expressionvectors when a yeast host cell such as Saccharomyces cerevisiae,Kluyveromyces lactis, or Pichia pastoris is used whereas fungaltranscription termination sequences would be used in host cells such asAspergillus niger, Neurospora crassa, or Tricoderma reesei.Transcription termination sequences include but are not limited to theSaccharomyces cerevisiae CYC transcription termination sequence (ScCYCTT), the Pichia pastoris ALG3 transcription termination sequence (ALG3TT), and Pichia pastoris PMA1 transcription termination sequence (PpPMA1TT).

The expression vectors of the present invention can also encodeselectable markers. Selectable markers are genetic functions that conferan identifiable trait upon a host cell so that cells transformed with avector carrying the selectable marker can be distinguished fromnon-transformed cells. Inclusion of a selectable marker into a vectorcan also be used to ensure that genetic functions linked to the markerare retained in the host cell population. Such selectable markers canconfer any easily identified dominant trait, e.g. drug resistance, theability to synthesize or metabolize cellular nutrients and the like.

Yeast selectable markers include drug resistance markers and geneticfunctions which allow the yeast host cell to synthesize essentialcellular nutrients, e.g. amino acids. Drug resistance markers which arecommonly used in yeast include chloramphenicol, kanamycin, methotrexate,G418 (geneticin), Zeocin, and the like. Genetic functions which allowthe yeast host cell to synthesize essential cellular nutrients are usedwith available yeast strains having auxotrophic mutations in thecorresponding genomic function. Common yeast selectable markers providegenetic functions for synthesizing leucine (LEU2), tryptophan (TRP1 andTRP2), uracil (URA3, URA5, URA6), histidine (HIS3), lysine (LYS2),adenine (ADE1 or ADE2), and the like. Other yeast selectable markersinclude the ARR3 gene from S. cerevisiae, which confers arseniteresistance to yeast cells that are grown in the presence of arsenite(Bobrowicz et al., Yeast, 13:819-828 (1997); Wysocki et al., J. Biol.Chem. 272:30061-066 (1997)). A number of suitable integration sitesinclude those enumerated in U.S. Published application No. 20070072262and include homologs to loci known for Saccharomyces cerevisiae andother yeast or fungi.

Therefore the present expression vectors can encode selectable markerswhich are useful for identifying and maintaining vector-containing hostcells within a cell population present in culture. In some circumstancesselectable markers can also be used to amplify the copy number of theexpression vector. After inducing transcription from the presentexpression vectors to produce an RNA encoding an overexpressed geneproduct or a heterologous chaperone protein, the RNA is translated bycellular factors to produce the gene product or the heterologouschaperone protein.

In yeast and other eukaryotes, translation of a messenger RNA (mRNA) isinitiated by ribosomal binding to the 5′ cap of the mRNA and migrationof the ribosome along the mRNA to the first AUG start codon wherepolypeptide synthesis can begin. Expression in yeast and mammalian cellsgenerally does not require specific number of nucleotides between aribosomal-binding site and an initiation codon, as is sometimes requiredin prokaryotic expression systems. However, for expression in a yeast ora mammalian host cell, the first AUG codon in an mRNA is preferably thedesired translational start codon.

Moreover, when expression is performed in a yeast host cell the presenceof long untranslated leader sequences, e.g. longer than 50-100nucleotides, can diminish translation of an mRNA. Yeast mRNA leadersequences have an average length of about 50 nucleotides, are rich inadenine, have little secondary structure and almost always use the firstAUG for initiation. Since leader sequences which do not have thesecharacteristics can decrease the efficiency of protein translation,yeast leader sequences are preferably used for expression of anoverexpressed gene product or a chaperone protein in a yeast host cell.The sequences of many yeast leader sequences are known and are availableto the skilled artisan, for example, by reference to Cigan et al. (Gene59: 1-18 (1987)).

In addition to the promoter, the ribosomal-binding site and the positionof the start codon, factors which can effect the level of expressionobtained include the copy number of a replicable expression vector. Thecopy number of a vector is generally determined by the vector's originof replication and any cis-acting control elements associated therewith.For example, an increase in copy number of a yeast episomal vectorencoding a regulated centromere can be achieved by inducingtranscription from a promoter which is closely juxtaposed to thecentromere. Moreover, encoding the yeast FLP function in a yeast vectorcan also increase the copy number of the vector.

One skilled in the art can also readily design and make expressionvectors which include the above-described sequences by combining DNAfragments from available vectors, by synthesizing nucleic acid moleculesencoding such regulatory elements or by cloning and placing newregulatory elements into the present vectors. Methods for makingexpression vectors are well-known. Overexpressed DNA methods are foundin any of the myriad of standard laboratory manuals on geneticengineering.

The expression vectors of the present invention can be made by ligatingthe heterologous chaperone protein coding regions in the properorientation to the promoter and other sequence elements being used tocontrol gene expression. After construction of the present expressionvectors, such vectors are transformed into host cells where theoverexpressed gene product and the heterologous chaperone protein can beexpressed. Methods for transforming yeast and other lower eukaryoticcells with expression vectors are well known and readily available tothe skilled artisan. For example, expression vectors can be transformedinto yeast cells by any of several procedures including lithium acetate,spheroplast, electroporation, and similar procedures.

Yeast host cells which can be used with yeast replicable expressionvectors include any wild type or mutant strain of yeast which is capableof secretion. Such strains can be derived from Saccharomyces cerevisiae,Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris,Schizosaccharomyces pombe, Yarrowia lipolytica, and related species ofyeast. In general, useful mutant strains of yeast include strains whichhave a genetic deficiency that can be used in combination with a yeastvector encoding a selectable marker. Many types of yeast strains areavailable from the Yeast Genetics Stock Center (Donner Laboratory,University of California, Berkeley, Calif. 94720), the American TypeCulture Collection (12301 Parklawn Drive, Rockville, Md. 20852,hereinafter ATCC), the National Collection of Yeast Cultures (FoodResearch Institute, Colney Lane, Norwich NR47UA, UK) and theCentraalbureau voor Schimmelcultures (Yeast Division, Julianalaan 67a,2628 BC Delft, Netherlands).

In general, lower eukaryotes such as yeast are useful for expression ofglycoproteins because they can be economically cultured, give highyields, and when appropriately modified are capable of suitableglycosylation. Yeast particularly offers established genetics allowingfor rapid transformations, tested protein localization strategies andfacile gene knock-out techniques. Suitable vectors have expressioncontrol sequences, such as promoters, including 3-phosphoglyceratekinase or other glycolytic enzymes, and an origin of replication,termination sequences and the like as desired.

Various yeasts, such as Kluyveromyces lactis, Pichia pastoris, Pichiamethanolica, and Hansenula polymorpha are useful for cell culturebecause they are able to grow to high cell densities and secrete largequantities of recombinant protein. Likewise, filamentous fungi, such asAspergillus niger, Fusarium sp, Neurospora crassa and others can be usedto produce glycoproteins of the invention at an industrial scale.

Lower eukaryotes, particularly yeast, can be genetically modified sothat they express glycoproteins in which the glycosylation pattern ishuman-like or humanized. Such can be achieved by eliminating selectedendogenous glycosylation enzymes and/or supplying exogenous enzymes asdescribed by Gerngross et al., US 20040018590. For example, a host cellcan be selected or engineered to be depleted in 1,6-mannosyl transferaseactivities, which would otherwise add mannose residues onto the N-glycanon a glycoprotein.

In one embodiment, the host cell further includes an α1,2-mannosidasecatalytic domain fused to a cellular targeting signal peptide notnormally associated with the catalytic domain and selected to target theα1,2-mannosidase activity to the ER or Golgi apparatus of the host cell.Passage of a recombinant glycoprotein through the ER or Golgi apparatusof the host cell produces a recombinant glycoprotein comprising aMan₅GlcNAc₂ glycoform, for example, a recombinant glycoproteincomposition comprising predominantly a Man₅GlcNAc₂ glycoform. Forexample, U.S. Pat. No. 7,029,872 and U.S. Published Patent ApplicationNos. 2004/0018590 and 2005/0170452 disclose lower eukaryote host cellscapable of producing a glycoprotein comprising a Man₅GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a GlcNAc transferase I (GnT I) catalytic domain fused to acellular targeting signal peptide not normally associated with thecatalytic domain and selected to target GlcNAc transferase I activity tothe ER or Golgi apparatus of the host cell. Passage of the recombinantglycoprotein through the ER or Golgi apparatus of the host cell producesa recombinant glycoprotein comprising a GlcNAcMan₅GlcNAc₂ glycoform, forexample a recombinant glycoprotein composition comprising predominantlya GlcNAcMan₅GlcNAc₂ glycoform. U.S. Pat. No. 7,029,872 and U.S.Published Patent Application Nos. 2004/0018590 and 2005/0170452 discloselower eukaryote host cells capable of producing a glycoproteincomprising a GlcNAcMan₅GlcNAc₂ glycoform. The glycoprotein produced inthe above cells can be treated in vitro with a hexaminidase to produce arecombinant glycoprotein comprising a Man₅GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a mannosidase II catalytic domain fused to a cellular targetingsignal peptide not normally associated with the catalytic domain andselected to target mannosidase II activity to the ER or Golgi apparatusof the host cell. Passage of the recombinant glycoprotein through the ERor Golgi apparatus of the host cell produces a recombinant glycoproteincomprising a GlcNAcMan₃GlcNAc₂ glycoform, for example a recombinantglycoprotein composition comprising predominantly a GlcNAcMan₃GlcNAc₂glycoform. U.S. Pat. No. 7,029,872 and U.S. Published Patent ApplicationNo. 2004/0230042 discloses lower eukaryote host cells that expressmannosidase II enzymes and are capable of producing glycoproteins havingpredominantly a GlcNAc₂Man₃GlcNAc₂ glycoform. The glycoprotein producedin the above cells can be treated in vitro with a hexaminidase toproduce a recombinant glycoprotein comprising a Man₃GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes GlcNAc transferase II (GnT II) catalytic domain fused to acellular targeting signal peptide not normally associated with thecatalytic domain and selected to target GlcNAc transferase II activityto the ER or Golgi apparatus of the host cell. Passage of therecombinant glycoprotein through the ER or Golgi apparatus of the hostcell produces a recombinant glycoprotein comprising a GlcNAc₂Man₃GlcNAc₂glycoform, for example a recombinant glycoprotein composition comprisingpredominantly a GlcNAc₂Man₃GlcNAc₂ glycoform. U.S. Pat. No. 7,029,872and U.S. Published Patent Application Nos. 2004/0018590 and 2005/0170452disclose lower eukaryote host cells capable of producing a glycoproteincomprising a GlcNAc₂Man₃GlcNAc₂ glycoform. The glycoprotein produced inthe above cells can be treated in vitro with a hexaminidase to produce arecombinant glycoprotein comprising a Man₃GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a galactosyltransferase catalytic domain fused to a cellulartargeting signal peptide not normally associated with the catalyticdomain and selected to target galactosyltransferase activity to the ERor Golgi apparatus of the host cell. Passage of the recombinantglycoprotein through the ER or Golgi apparatus of the host cell producesa recombinant glycoprotein comprising a GalGlcNAc₂Man₃GlcNAc₂ orGal₂GlcNAc₂Man₃GlcNAc₂ glycoform, or mixture thereof for example arecombinant glycoprotein composition comprising predominantly aGalGlcNAc₂Man₃GlcNAc₂ glycoform or Gal₂GlcNAc₂Man₃GlcNAc₂ glycoform ormixture thereof. U.S. Pat. No. 7,029,872 and U.S. Published PatentApplication No. 2006/0040353 discloses lower eukaryote host cellscapable of producing a glycoprotein comprising a Gal₂GlcNAc₂Man₃GlcNAc₂glycoform. The glycoprotein produced in the above cells can be treatedin vitro with a galactosidase to produce a recombinant glycoproteincomprising a GlcNAc₂Man₃GlcNAc₂ glycoform, for example a recombinantglycoprotein composition comprising predominantly a GlcNAc₂Man₃GlcNAc₂glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a sialyltransferase catalytic domain fused to a cellulartargeting signal peptide not normally associated with the catalyticdomain and selected to target sialytransferase activity to the ER orGolgi apparatus of the host cell. Passage of the recombinantglycoprotein through the ER or Golgi apparatus of the host cell producesa recombinant glycoprotein comprising predominantly aNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ glycoform or NANAGal₂GlcNAc₂Man₃GlcNAc₂glycoform or mixture thereof. For lower eukaryote host cells such asyeast and filamentous fungi, it is useful that the host cell furtherinclude a means for providing CMP-sialic acid for transfer to theN-glycan. U.S. Published Patent Application No. 2005/0260729 discloses amethod for genetically engineering lower eukaryotes to have a CMP-sialicacid synthesis pathway and U.S. Published Patent Application No.2006/0286637 discloses a method for genetically engineering lowereukaryotes to produce sialylated glycoproteins. The glycoproteinproduced in the above cells can be treated in vitro with a neuraminidaseto produce a recombinant glycoprotein comprising predominantly aGal₂GlcNAc₂Man₃GlcNAc₂ glycoform or GalGlcNAc₂Man₃GlcNAc₂ glycoform ormixture thereof.

Any one of the preceding host cells can further include one or moreGlcNAc transferase selected from the group consisting of GnT III, GnTIV, GnT V, GnT VI, and GnT IX to produce glycoproteins having bisected(GnT III) and/or multiantennary (GnT IV, V, VI, and IX) N-glycanstructures such as disclosed in U.S. Published Patent Application Nos.2004/074458 and 2007/0037248.

In further embodiments, the host cell that produces glycoproteins thathave predominantly GlcNAcMan₅GlcNAc₂ N-glycans further includes agalactosyltransferase catalytic domain fused to a cellular targetingsignal peptide not normally associated with the catalytic domain andselected to target Galactosyltransferase activity to the ER or Golgiapparatus of the host cell. Passage of the recombinant glycoproteinthrough the ER or Golgi apparatus of the host cell produces arecombinant glycoprotein comprising predominantly theGalGlcNAcMan₅GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell thatproduced glycoproteins that have predominantly the predominantly theGalGlcNAcMan₅GlcNAc₂ N-glycans further includes a sialyltransferasecatalytic domain fused to a cellular targeting signal peptide notnormally associated with the catalytic domain and selected to targetsialytransferase activity to the ER or Golgi apparatus of the host cell.Passage of the recombinant glycoprotein through the ER or Golgiapparatus of the host cell produces a recombinant glycoproteincomprising a NANAGalGlcNAcMan₅GlcNAc₂ glycoform.

Various of the preceding host cells further include one or more sugartransporters such as UDP-GlcNAc transporters (for example, Kluyveromyceslactis and Mus musculus UDP-GlcNAc transporters), UDP-galactosetransporters (for example, Drosophila melanogaster UDP-galactosetransporter), and CMP-sialic acid transporter (for example, human sialicacid transporter). Because lower eukaryote host cells such as yeast andfilamentous fungi lack the above transporters, it is preferable thatlower eukaryote host cells such as yeast and filamentous fungi begenetically engineered to include the above transporters.

In further embodiments of the above host cells, the host cells arefurther genetically engineered to eliminate glycoproteins havinga-mannosidase-resistant N-glycans by deleting or disrupting the3-mannosyltransferase gene (BMT2) (See, U.S. Published PatentApplication No. 2006/0211085) and glycoproteins having phosphomannoseresidues by deleting or disrupting one or both of the phosphomannosyltransferase genes PNO1 and MNN4B (See for example, U.S. Pat. Nos.7,198,921 and 7,259,007). In further still embodiments of the above hostcells, the host cells are further genetically modified to eliminateO-glycosylation of the glycoprotein by deleting or disrupting one ormore of the protein O-mannosyltransferase (Dol-P-Man:Protein (Ser/Thr)Mannosyl Transferase genes) (PMTs) (See U.S. Pat. No. 5,714,377) orgrown in the presence of i inhibitors such as Pmt-1, Pmti-2, and Pmti-3as disclosed in Published International Application No. WO 2007061631,or both.

Thus, provided are host cells that have been genetically modified toproduce glycoproteins wherein the predominant N-glycans thereon includebut are not limited to Man₈GlcNAc₂, Man₇GlcNAc₂, Man₆GlcNAc₂,Man₅GlcNAc₂, GlcNAcMan₅GlcNAc₂, GalGlcNAcMan₅GlcNAc₂,NANAGalGlcNAcMan₅GlcNAc₂, Man₃GlcNAc₂, GlcNAc₍₁₋₄₎Man₃GlcNAc₂,Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂, NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂.Further included are host cells that produce glycoproteins that haveparticular mixtures of the aforementioned N-glycans thereon.

In the following examples, heterologous human proteins are expressed inhost cells of the species Pichia pastoris. These examples demonstratethe invention with respect to specific embodiments of the invention, andare not to be construed as limiting in any manner. The skilled artisan,having read the disclosure and examples herein, will recognize thatnumerous variants, modifications and improvements to the methods andmaterials described that are possible without deviating from thepractice of the present invention.

Example 1

This example shows that expression of heterologous human proteins inPichia pastoris was enhanced by using host cells in which the geneencoding the endogenous PDI1 has been inactivated and replaced with anexpression cassette encoding the human PDI. The example further showsthat these host cells produced recombinant antibodies that had reducedO-glycosylation.

Construction of expression/integration plasmid vector pGLY642 comprisingan expression cassette encoding the human PDI protein and nucleic acidmolecules to target the plasmid vector to the Pichia pastoris PDI1 locusfor replacement of the gene encoding the Pichia pastoris PDI1 with anucleic acid molecule encoding the human PDI was as follows and is shownin FIG. 8. cDNA encoding the human PDI was amplified by PCR using theprimers hPDI/UP1: 5′ AGCGCTGACGCCCCCGAGGAGGAGGACCAC 3′ (SEQ ID NO: 1)and hPDI/LP-PacI: 5′ CCTTAATTAATTACAGTTCATCATGCACAGCTTTCTGATCAT 3′ (SEQID NO: 2), Pfu turbo DNA polymerase (Stratagene, La Jolla, Calif.), anda human liver cDNA (BD Bioscience, San Jose, Calif.). The PCR conditionswere 1 cycle of 95° C. for two minutes, 25 cycles of 95° C. for 20seconds, 58° C. for 30 seconds, and 72° C. for 1.5 minutes, and followedby one cycle of 72° C. for 10 minutes. The resulting PCR product wascloned into plasmid vector pCR2.1 to make plasmid vector pGLY618. Thenucleotide and amino acid sequences of the human PDI (SEQ ID NOs: 39 and40, respectively) are shown in Table 11.

The nucleotide and amino acid sequences of the Pichia pastoris PDI1 (SEQID NOs:41 and 42, respectively) are shown in Table 11. Isolation ofnucleic acid molecules comprising the Pichia pastoris PDI1 5′ and 3′regions was performed by PCR amplification of the regions from Pichiapastoris genomic DNA. The 5′ region was amplified using primers PB248:5′ ATGAATTCAGGCCATATCGGCCATTGTTTACTGTGCGCCCACAGT AG 3′ (SEQ ID NO: 3);PB249: 5′ ATGTTTAAACGTGAGGATTACTGGTGATGAAAGAC 3′ (SEQ ID NO: 4). The 3′region was amplified using primers PB250: 5′AGACTAGTCTATTTGGAGACATTGACGGATCCAC 3′ (SEQ ID NO: 5); PB251: 5′ATCTCGAGAGGCCATGCAGGCCAACCACAAGATGAATCAAATTTTG-3′ (SEQ ID NO: 6). Pichiapastoris strain NRRL-Y11430 genomic DNA was used for PCR amplification.The PCR conditions were one cycle of 95° C. for two minutes, 25 cyclesof 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 2.5minutes, and followed by one cycle of 72° C. for 10 minutes. Theresulting PCR fragments, PpPDI1 (5′) and PpPDI1 (3′), were separatelycloned into plasmid vector pCR2.1 to make plasmid vectors pGLY620 andpGLY617, respectively. To construct pGLY678, DNA fragments PpARG3-5′ andPpARG-3′ of integration plasmid vector pGLY24, which targets the plasmidvector to Pichia pastoris ARG3 locus, were replaced with DNA fragmentsPpPDI (5) and PpPDI (3D, respectively, which targets the plasmid vectorpGLY678 to the PDI1 locus and disrupts expression of the PDI1 locus.

The nucleic acid molecule encoding the human PDI was then cloned intoplasmid vector pGLY678 to produce plasmid vector pGLY642 in which thenucleic acid molecule encoding the human PDI was placed under thecontrol of the Pichia pastoris GAPDH promoter (PpGAPDH).Expression/integration plasmid vector pGLY642 was constructed byligating a nucleic acid molecule (SEQ ID NO: 27) encoding theSaccharomyces cerevisiae alpha mating factor pre-signal peptide(ScaMFpre-signal peptide (SEQ ID NO: 28) having a NotI restrictionenzyme site at the 5′ end and a blunt 3′ end and the expression cassettecomprising the nucleic acid molecule encoding the human PDI releasedfrom plasmid vector pGLY618 with AfeI and PacI to produce a nucleic acidmolecule having a blunt 5′ end and a PacI site at the 3′ end intoplasmid vector pGLY678 digested with NotI and Pad. The resultingintegration/expression plasmid vector pGLY642 comprises an expressioncassette encoding a human PDI/ScaMFpre-signal peptide fusion proteinoperably linked to the Pichia pastoris promoter and nucleic acidmolecule sequences to target the plasmid vector to the Pichia pastorisPDI1 locus for disruption of the PDI1 locus and integration of theexpression cassette into the PDI1 locus. FIG. 8 illustrates theconstruction of plasmid vector pGLY642. The nucleotide and amino acidsequences of the ScaMFpre-signal peptide are shown in SEQ ID NOs: 27 and28, respectively.

Construction of expression/integration vector pGLY2232 encoding thehuman ERO1α protein was as follows and is shown in FIG. 9. A nucleicacid molecule encoding the human ERO1α protein was synthesized byGeneArt AG (Regensburg, Germany) and used to construct plasmid vectorpGLY2224. The nucleotide and amino acid sequences of the human ERO1αprotein (SEQ ID NOs: 43 and 44, respectively) are shown in Table 11. Thenucleic acid molecule encoding the human ERO1α protein was released fromthe plasmid vector using restriction enzymes AfeI and FseI and thenligated with a nucleic acid molecule encoding the ScaMPpre-signalpeptide with 5′ NotI and 3′ blunt ends as above into plasmid vectorpGLY2228 digested with NotI and FseI. Plasmid vector pGLY2228 alsoincluded nucleic acid molecules that included the 5′ and 3′ regions ofthe Pichia pastoris PRB1 gene (PpPRB1-5′ and PpPRB1-3′ regions,respectively). The resulting plasmid vector, pGLY2230 was digested withBglII and NotI and then ligated with a nucleic acid molecule containingthe Pichia pastoris PDI1 promoter (PpPDI promoter) which had beenobtained from plasmid vector pGLY2187 digested with BglII and NotI. Thenucleotide sequence of the PpPDI promoter is 5′-AACACGAACACTGTAAATAGAATAAAAGAAAACTTGGATAGTAGAACTTCAATGTAGTGTTTCTATTGTCTTACGCGGCTCTTTAGATTGCAATCCCCAGAATGGAATCGTCCATCTTTCTCAACCCACTCAAAGATAATCTACCAGACATACCTACGCCCTCCATCCCAGCACCACGTCGCGATCACCCCTAAAACTTCAATAATTGAACACGTACTGATTTCCAAACCTTCTTCTTCTTCCTATCTATAAGA-3′(SEQ ID NO: 59). The resulting plasmid vector, pGLY2232, is anexpression/integration vector that contains an expression cassette thatencodes the human ERO1α fusion protein under control of the Pichiapastoris PDI1 promoter and includes the 5′ and 3′ regions of the Pichiapastoris PRB1 gene to target the plasmid vector to the PRB1 locus ofgenome for disruption of the PRB1 locus and integration of theexpression cassette into the PRB1 locus. FIG. 9 illustrates theconstruction of plasmid vector pGLY2232.

Construction of expression/integration vector pGLY2233 encoding thehuman GRP94 protein was as follows and is shown in FIG. 10. The humanGRP94 was PCR amplified from human liver cDNA (BD Bioscience) with theprimers hGRP94/UP1: 5′-AGCGCTGACGATGAAGTTGATGTGGATGGTACAGTAG-3; (SEQ IDNO: 15); and hGRP94/LP1: 5′-GGCCG GCCTT ACAAT TCATC ATGTT CAGCT GTAGATTC 3; (SEQ ID NO: 16). The PCR conditions were one cycle of 95° C. fortwo minutes, 25 cycles of 95° C. for 20 seconds, 55° C. for 20 seconds,and 72° C. for 2.5 minutes, and followed by one cycle of 72° C. for 10minutes. The PCR product was cloned into plasmid vector pCR2.1 to makeplasmid vector pGLY2216. The nucleotide and amino acid sequences of thehuman GRP94 (SEQ ID NOs: 45 and 46, respectively) are shown in Table 11.

The nucleic acid molecule encoding the human GRP94 was released fromplasmid vector pGLY2216 with AfeI and FseI. The nucleic acid moleculewas then ligated to a nucleic acid molecule encoding the ScaMPpre-signalpeptide having NotI and blunt ends as above and plasmid vector pGLY2231digested with NotI and FseI carrying nucleic acid molecules comprisingthe Pichia pastoris PEP4 5′ and 3′ regions (PpPEP4-5′ and PpPEP4-3′regions, respectively) to make plasmid vector pGLY2229. Plasmid vectorpGLY2229 was digested with BglII and NotI and a DNA fragment containingthe PpPDI1 promoter was removed from plasmid vector pGLY2187 with BglIIand NotI and the DNA fragment ligated into pGLY2229 to make plasmidvector pGLY2233. Plasmid vector pGLY2233 encodes the human GRP94 fusionprotein under control of the Pichia pastoris PDI promoter and includesthe 5′ and 3′ regions of the Pichia pastoris PEP4 gene to target theplasmid vector to the PEP4 locus of genome for disruption of the PEP4locus and integration of the expression cassette into the PEP4 locus.FIG. 10 illustrates the construction of plasmid vector pGLY2233.

Construction of plasmid vectors pGLY1162, pGLY1896, and pGFI207t was asfollows. All Trichoderma reesei α-1,2-mannosidase expression plasmidvectors were derived from pGFI165, which encodes the T. reeseiα-1,2-mannosidase catalytic domain (See published InternationalApplication No. WO2007061631) fused to S. cerevisiae αMATpre signalpeptide herein expression is under the control of the Pichia pastorisGAP promoter and wherein integration of the plasmid vectors is targetedto the Pichia pastoris PRO1 locus and selection is using the Pichiapastoris URA5 gene. A map of plasmid vector pGFI165 is shown in FIG. 11.

Plasmid vector pGLY1162 was made by replacing the GAP promoter inpGFI165 with the Pichia pastoris AOX1 (PpAOX1) promoter. This wasaccomplished by isolating the PpAOX1 promoter as an EcoRI (madeblunt)-BglII fragment from pGLY2028, and inserting into pGFI165 that wasdigested with NotI (made blunt) and BglII. Integration of the plasmidvector is to the Pichia pastoris PRO1 locus and selection is using thePichia pastoris URA5 gene. A map of plasmid vector pGLY1162 is shown inFIG. 12.

Plasmid vector pGLY1896 contains an expression cassette encoding themouse α-1,2-mannosidase catalytic domain fused to the S. cerevisiae MNN2membrane insertion leader peptide fusion protein (See Choi at al., Proc.Natl. Acad. Sci. USA 100: 5022 (2003)) inserted into plasmid vectorpGFI165 (FIG. 12). This was accomplished by isolating theGAPp-ScMNN2-mouse MNSI expression cassette from pGLY1433 digested withXhoI (and the ends made blunt) and PmeI, and inserting the fragment intopGFI165 that digested with PmeI. Integration of the plasmid vector is tothe Pichia pastoris PRO1 locus and selection is using the Pichiapastoris URA5 gene. A map of plasmid vector pGLY1896 is shown in FIG.11.

Plasmid vector pGFI207t is similar to pGLY1896 except that the URA5selection marker was replaced with the S. cerevisiae ARR3 (ScARR3) gene,which confers resistance to arsenite. This was accomplished by isolatingthe ScARR3 gene from pGFI166 digested with AscI and the AscI ends madeblunt) and BglII, and inserting the fragment into pGLY1896 that digestedwith SpeI and the SpeI ends made blunt and BglII. Integration of theplasmid vector is to the Pichia pastoris PRO1 locus and selection isusing the Saccharomyces cerevisiae ARR3 gene. A map of plasmid vectorpGFI207t is shown in FIG. 11.

Construction of anti-DKK1 antibody expression/integration plasmidvectors pGLY2260 and pGLY2261 was as follows. Anti-DKK1 antibodies areantibodies that recognize Dickkopf protein 1, a ligand involved in theWnt signaling pathway. To generate expression/integration plasmidvectors pGLY2260 and pGLY2261 encoding an anti-DKK1 antibody,codon-optimized nucleic acid molecules encoding heavy chain (HC; fusionprotein containing VH+IgG₂m4) and light chain (LC; fusion proteincontaining VL+Lλ. constant region) fusion proteins, each in frame with anucleic acid molecule encoding an α-amylase (from Aspergillus niger)signal peptide were synthesized by GeneArt AG. The nucleotide and aminoacid sequences for the α-amylase signal peptide are shown in SEQ ID NOs:33 and 34. The nucleotide sequence of the HC is shown in SEQ ID NO: 51and the amino acid sequence is shown in SEQ ID NO: 52. The nucleotidesequence of the LC is shown in SEQ ID NO: 53 and the amino acid sequenceis shown in SEQ ID NO: 54. The IgG₂ m4 isotype has been disclosed inU.S. Published Application No. 2007/0148167 and U.S. PublishedApplication No. 2006/0228349. The nucleic acid molecules encoding the HCand LC fusion proteins were separately cloned using unique 5′-EcoRI and3′-FseI sites into expression plasmid vector pGLY1508 to form plasmidvectors pGLY1278 and pGLY1274, respectively. These plasmid vectorscontained the Zeocin-resistance marker and TRP2 integration sites andthe Pichia pastoris AOX1 promoter operably linked to the nucleic acidmolecules encoding the HC and LC fusion proteins. The LC fusion proteinexpression cassette was removed from pGLY1274 with BglII and BamH1 andcloned into pGLY1278 digested with BglII to generate plasmid vectorpGLY2260, which encodes the HC and LC fusion proteins and targets theexpression cassettes to the TRP2 locus for integration of the expressioncassettes into the TRP2 locus. The plasmid vector pGLY2261 contains anadditional LC in plasmid vector pGLY2260. (FIG. 13).

Construction of anti-ADDL antibody expression/integration plasmid vectorpGLY2260 was as follows. Anti-ADDL antibodies are antibodies thatrecognize An-derived diffusible ligands, see for example U.S. PublishedApplication No. 20070081998. To generate expression/integration plasmidvector pGLY2012, codon-optimized nucleic acid molecules encoding heavychain (HC; contained VH+IgG2m4) and light chain (LC; fusion proteincontaining VL+Lλ, constant region) fusion proteins, each in frame with anucleic acid molecule encoding Saccharomyces cerevisiae invertase signalpeptide were synthesized by GeneArt AG. The nucleic acid moleculesencoding the HC and LC fusion proteins were separately cloned usingunique 5′-EcoRI and 3′-FseI sites into expression/integration plasmidvectors pGLY1508 and pGLY1261 to form pGLY2011 and pGLY2010,respectively, which contained the Zeocin-resistance marker and TRP2integration sites and the Pichia pastoris AOX1 promoter operably linkedto the nucleic acid molecules encoding the HC and LC fusion proteins.The HC expression cassette was removed from pGLY2011 with BglII and NotIand cloned into pGLY2010 digested with BamHI and NotI to generatepGLY2012, which encodes the HC and LC fusion proteins and targets theexpression cassettes to the TRP2 locus for integration of the expressioncassettes into the TRP2 locus (FIG. 14).

Yeast transformations with the above expression/integration vectors wereas follows. Pichia pastoris strains were grown in 50 mL YPD media (yeastextract (1%), peptone (2%), dextrose (2%)) overnight to an OD of betweenabout 0.2 to 6.0. After incubation on ice for 30 minutes, cells werepelleted by centrifugation at 2500-3000 rpm for 5 minutes. Media wasremoved and the cells washed three times with ice cold sterile 1Msorbitol before resuspension in 0.5 ml ice cold sterile 1M sorbitol. TenμL linearized DNA (5-20 μg) and 100 μL cell suspension was combined inan electroporation cuvette and incubated for 5 minutes on ice.Electroporation was in a Bio-Rad GenePulser Xcell following the presetPichia pastoris protocol (2 kV, 25 μF, 200Ω), immediately followed bythe addition of 1 mL YPDS recovery media (YPD media plus 1 M sorbitol).The transformed cells were allowed to recover for four hours toovernight at room temperature (24° C.) before plating the cells onselective media.

Generation of Cell Lines was as follows and is shown in FIG. 3. Thestrain yGLY24-1 (ura5Δ::MET1 ochIΔ::lacZbmt2Δ::lacZ/KlMNN2-2/mnn4L1Δ::lacZ/MmSLC35A3 pno1Δmnn4Δ:lacZmet16Δ::lacZ), was constructed using methods described earlier (See forexample, Nett and Gerngross, Yeast 20:1279 (2003); Choi et al., Proc.Natl. Acad. Sci. USA 100:5022 (2003); Hamilton et al., Science 301:1244(2003)). The BMT2 gene has been disclosed in Mille et al., J. Biol.Chem. 283: 9724-9736 (2008) and U.S. Published Application No.20060211085. The PNO1 gene has been disclosed in U.S. Pat. No. 7,198,921and the mnn4L1 gene (also referred to as mnn4b) has been disclosed inU.S. Pat. No. 7,259,007. The mnn4 refers to mnn4L2 or mnn4a. In thegenotype, KlMNN2-2 is the Kluveromyces lactis GlcNAc transporter andMmSLC35A3 is the Mus musculus GlcNAc transporter. The URA5 deletionrenders the yGLY24-1 strain auxotrophic for uracil (See U.S. Publishedapplication No. 2004/0229306) and was used to construct the humanizedchaperone strains that follow. While the various expression cassetteswere integrated into particular loci of the Pichia pastoris genome inthe examples herein, it is understood that the operation of theinvention is independent of the loci used for integration. Loci otherthan those disclosed herein can be used for integration of theexpression cassettes. Suitable integration sites include thoseenumerated in U.S. Published application No. 20070072262 and includehomologs to loci known for Saccharomyces cerevisiae and other yeast orfungi.

Control strain yGLY645 (PpPDI1) was constructed. Strain yGLY645expresses both a Trichoderma Reesei mannosidase1 (TrMNS1) and a mousemannosidase IA (MuMNS1A), each constitutively expressed under thecontrol of a PpGAPDH promoter, with the native Pichia pastoris PDI1locus intact. Strain yGLY645 was generated from strain yGLY24-1 bytransforming yGLY24-1 with plasmid vector pGLY1896, which targeted theplasmid vector to the Proline 1 (PRO1) locus in the Pichia genome.Plasmid vector pGLY1896 contains expression cassettes encoding theTrichoderma Reesei mannosidase 1 (TrMNS1) and the mouse mannosidase IA(FB53, MuMNS1A), each constitutively expressed under the control of aPpGAPDH promoter.

Strains yGLY702 and yGLY704 were generated in order to test theeffectiveness of the human PDI1 expressed in Pichia pastoris cells inthe absence of the endogenous Pichia pastoris PDI1 gene. Strains yGLY702and yGLY704 (hPDI) were constructed as follows. Strain yGLY702 wasgenerated by transforming yGLY24-1 with plasmid vector pGLY642containing the expression cassette encoding the human PDI under controlof the constitutive PpGAPDH promoter. Plasmid vector pGLY642 alsocontained an expression cassette encoding the Pichia pastoris URA5,which rendered strain yGLY702 prototrophic for uracil. The URA5expression cassette was removed by counterselecting yGLY702 on 5-FOAplates to produce strain yGLY704 in which, so that the Pichia pastorisPDI1 gene has been stably replaced by the human PDI gene and the strainis auxotrophic for uracil.

The replacement of the Pichia pastoris PDI1 with the human PDI usingplasmid vector pGLY642 was confirmed by colony PCR using the followingprimers specific to only the PpPDI1 ORF; PpPDI/UPi-1,5′-GGTGAGGTTGAGGTCCCAAGTGACTATCAAGGTC-3; (SEQ ID NO: 7); PpPDI/LPi-1,5′-GACCTTGATAGTCACTTGGGACCTCAACCTCACC-3; (SEQ ID NO: 8); PpPDI/UPi-2, 5′CGCCAATGATGAGGATGCCTCTTCAAAGGT TGTG-3; (SEQ ID NO: 9); and PpPDI/LPi-2,5′-CACAACCTTTGAAGAGGCATCCTCATCATTGGCG-3; (SEQ ID NO: 10). Thus, theabsence of PCR product indicates the knockout of PpPDI1. The PCRconditions were one cycle of 95° C. for two minutes, 25 cycles of 95° C.for 20 seconds, 58° C. for 20 seconds, and 72° C. for one minute, andfollowed by one cycle of 72° C. for 10 minutes.

Additional PCR was used to confirm the double crossover of pGLY642 atthe PpPDI1 locus using PCR primers; PpPDI-5′/UP,5′-GGCGATTGCATTCGCGACTGTATC-3; (SEQ ID NO: 11); and, hPDI-3′/LP5′-CCTAGAGAGCGOTGGCCAAGATG-3; (SEQ ID NO: 12). PpPDI-5′/UP primes theupstream region of PpPDI1 that is absent in PpPDI1 (5′) of pGY642 andhPDI-3′/LP primes human PDI ORF in pGLY642. The PCR conditions were onecycle of 95° C. for two minutes, 25 cycles of 95° C. for 20 seconds, 50°C. for 30 seconds, and 72° C. for 2.5 minutes, and followed by one cycleof 72° C. for 10 minutes.

The integration efficiency of a plasmid vector as a knockout (i.e., adouble cross-over event) or as a ‘roll-in’ (i.e., a single integrationof the plasmid vector into the genome, can be dependent upon a number offactors, including the number and length of homologous regions betweenvectors and the corresponding genes on host chromosomal DNA, selectionmarkers, the role of the gene of interest, and the ability of theknocked-in gene to complement the endogenous function. The inventorsfound that in some instances pGLY642 was integrated as a doublecross-over, resulting in replacement of the endogenous PpPDI gene withhuman PpPDI, while in other cases, the pGLY642 plasmid vector wasintegrated as a single integration, resulting in presence of both theendogenous PpPDI1 gene and a human PpPDI gene. In order to distinguishbetween these events, the inventors utilized PCR primers of Sequence IDNos. 11 through 14, described herein. If the PpPDI gene has beenretained after integration of the pGLY642 plasmid vector, PpPDI-5′/UPand hPDI-3′/LP, directed to the internal PpPDI coding sequence, willresult in an amplification product and a corresponding band. In theevent of a knockout or double cross-over, these primers will not resultin any amplification product and no corresponding band will be visible.

The roll-in of pGLY642 was confirmed with the primers; PpPDI/UPi (SEQ IDNO: 7) and PpPDI/LPi-1 (SEQ ID NO: 8) encoding PpPDI1, and hPDI/UP,5′-GTGGCCACACCAGGGGGCATGGAAC-3; (SEQ ID NO: 13); and hPDI-3′/LP,5′-CCTAGAGAGCGGTGGCCAAG ATG-3; (SEQ ID NO: 14); encoding human PDI. ThePCR conditions were one cycle of 95° C. for two minutes, 25 cycles of95° C. for 20 seconds, 58° C. for 20 seconds, and 72° C. for one minute,and followed by 1 cycle of 72° C. for 10 minutes for PpPDI1, and 1 cycleof 95° C. for two minutes, 25 cycles of 95° C. for 20 seconds, 50° C.for 30 seconds, and 72° C. for 2.5 minutes, and followed by one cycle of72° C. for 10 minutes for human PDI.

Strain yGLY714 is a strain that contains both the Pichia pastoris PDI1locus and expresses the human PDI and was a result of integration via asingle crossover event. Strain yGLY714 was generated from strainyGLY24-1 by integrating plasmid vector pGLY642, which comprises thehuman PDI gene under constitutive regulatory control of the Pichiapastoris GAPDH promoter, into the PpPDI 5′UTR region in yGLY24-1.Integration of this vector does not disrupt expression of the Pichiapastoris PDI1 locus. Thus, in yGLY714, the human PDI is constitutivelyexpressed in the presence of the Pichia pastoris endogenous PDI1.

Strain yGLY733 was generated by transforming with plasmid vectorpGLY1162, which comprises an expression cassette that encodes theTrichoderma Reesei mannosidase (TrMNS1) operably linked to the Pichiapastoris AOX1 promoter (PpAOX1-TrMNS1), into the PRO1 locus of yGLY704.This strain has the gene encoding the Pichia pastoris PD1 replaced withthe expression cassette encoding the human PDI1, has the PpAOX1-TrMNS1expression cassette integrated into the PRO1 locus, and is a URA5prototroph. The PpAOX1 promoter allows overexpression when the cells aregrown in the presence of methanol.

Strain yGLY762 was constructed by integrating expression cassettesencoding TrMNS1 and mouse mannosidase IA (MuMNS1A), each operably linkedto the Pichia pastoris GAPDH promoter in plasmid vector pGFI207t intostrain yGLY733 at the 5′ PRO1 locus UTR in Pichia pastoris genome. Thisstrain has the gene encoding the Pichia pastoris PDI1 replaced with theexpression cassette encoding the human PDI, has the PpGAPDH-TrMNS1 andPpGAPDH-MuMNS1A expression cassettes integrated into the PRO1 locus, andis a URA5 prototroph.

Strain yGLY730 is a control strain for strain yGLY733. Strain yGLY730was generated by transforming pGLY1162, which comprises an expressioncassette that encodes the Trichoderma Reesei mannosidase (TrMNS1)operably linked to the Pichia pastoris AOX1 promoter (PpAOX1-TrMNS1),into the PRO1 locus of yGLY24-1. This strain has the Pichia pastorisPDI1, has the PpAOX1-TrMNS1 expression cassette integrated into the PRO1locus, and is a URA5 prototroph.

Control Strain yGLY760 was constructed by integrating expressioncassettes encoding TrMNS1 and mouse mannosidase IA (MuMNS1A), eachoperably linked to the Pichia pastoris GAPDH promoter in plasmid vectorpGFI207t into control strain yGLY730 at the 5′ PRO1 locus UTR in Pichiapastoris genome. This strain has the gene encoding the Pichia pastorisPDI1, has the PpGAPDH-TrMNS1 and PpGAPDH-MuMNS1A expression cassettesintegrated into the PRO1 locus, and is a URA5 prototroph.

Strain yGLY2263 was generated by transforming strain yGLY645 withintegration/expression plasmid pGLY2260, which targets an expressioncassette encoding the anti-DKK1 antibody to the TRP2 locus.

Strain yGLY2674 was generated by counterselecting yGLY733 on 5-FOAplates. This strain has the gene encoding the Pichia pastoris PDI1replaced with the expression cassette encoding the human PDI, has thePpAOX1-TrMNS1 expression cassette integrated into the PRO1 locus, and isa URA5 auxotroph.

Strain yGLY2677 was generated by counterselecting yGLY762 on 5-FOAplates. This strain has the gene encoding the Pichia pastoris PDI1replaced with the expression cassette encoding the human PDI, has thePpAOX1-TrMNS1 expression cassette integrated into the PRO1 locus, hasthe PpGAPH-TrMNS1 and PpGAPDH-MuMNS1A expression cassettes integratedinto the PRO1 locus, and is a URA5 auxotroph.

Strains yGLY2690 was generated by integrating plasmid vector pGLY2232,which encodes the human ERO1α protein, into the PRB1 locus. This strainhas the gene encoding the Pichia pastoris PDI1 replaced with theexpression cassette encoding the human PDI, has the PpAOX1-TrMNS1expression cassette integrated into the PRO1 locus, the human ERO1αexpression cassette integrated into the PRB1 locus, and is a URA5prototroph.

Strains yGLY2696 was generated by integrating plasmid vector pGLY2233,which encodes the human GRP94 protein, into the PEP4 locus. This strainhas the gene encoding the Pichia pastoris PDI1 replaced with theexpression cassette encoding the human PDI, has the PpAOX1-TrMNS1expression cassette integrated into the PRO1 locus, has thePpGAPDH-TrMNS1 and PpGAPDH-MuMNS1A expression cassettes integrated intothe PRO1 locus, has the human GRP94 integrated into the PEP4 locus, andis a URA5 prototroph.

Strain yGLY3628 was generated by transforming strain yGLY2696 withintegration/expression plasmid pGLY2261, which targets an expressioncassette encoding the anti-DKK1 antibody to the TRP2 locus.

Strain yGLY3647 was generated by transforming strain yGLY2690 withintegration/expression plasmid pGLY2261, which targets an expressioncassette encoding the anti-DKK1 antibody to the TRP2 locus.

The yield of protein produced in a strain, which expresses the human PDIprotein in place of the Pichia pastoris PDI1 protein, was compared tothe yield of the same protein produced in a strain, which expresses boththe human and Pichia pastoris PDI proteins, and a strain, whichexpresses only the Pichia pastoris PDI1 protein. Strain yGLY733, whichexpresses the human PDI protein in place of the Pichia pastoris PDI1protein, strain yGLY714, which expresses both the human and Pichiapastoris PDI1 proteins, and strain yGLY730, which expresses only thePichia pastoris PDI1 protein were evaluated to determine the effect ofreplacing the Pichia pastoris PDI1 protein with the human PDI protein onantibody titers produced by the strains. All three yeast strains weretransformed with plasmid vector pGLY2261, which encodes the anti-DKK1antibody.

Titer improvement for culture growth was determined from deep-well platescreening in accordance with the NIH ImageJ software protocol, asdescribed in Rasband, ImageJ, U.S. National Institutes of Health,Bethesda, Md., USA, 1997-2007; and Abramoff, et al., BiophotonicsInternational, 11: 36-42 (2004). Briefly, antibody screening in 96deep-well plates was performed essentially as follows. Transformantswere inoculated to 600 μL BMGY and grown at 24° C. at 840 rpm for twodays in a Micro-Plate Shaker. The resulting 50 μL seed culture wastransferred to two 96-well plates containing 600 μL fresh BMGY per welland incubated for two days at the same culture condition as above. Thetwo expansion plates were combined to one prior to centrifugation for 5minutes at 1000 rpm, the cell pellets were induced in 600 μL BMMY perwell for two days and then the centrifuged 400 μL clear supernatant waspurified using protein A beads. The purified proteins were subjected toSDS-PAGE electrophoresis and the density of protein bands were analyzedusing NIH ImageJ software.

Representative results are shown in FIG. 1. FIG. 1 (Panel B) shows thatwhile yGLY714, which expresses both Pichia pastoris PDI1 and human PDI,improved yield two-fold over the control (yGLY730) (Panel A), afive-fold increase in yield was achieved with strain yGLY733, whichexpresses only the human PDI (Panel C). The results are also presentedin Table 1.

TABLE 1 Replacement of PpPDI1 yGLY714 yGLY730 (Both Pichia and yGLY733(control) human PDI) (human PDI) Pichia pastoris PDI1 Wild-typeWild-type Knockout Human PDI None Overexpression Overexpression Titerimprovement Control 2-fold 5-fold

Strains yGLY730 and yGLY733 were transformed with plasmid vectorpGLY2012 which encodes the anti-ADDL antibody. The transformed strainswere evaluated by 96 deep well screening as described above and antibodywas produced in 500 mL SixFors and 3 L fermentors using the followingprocedures. Bioreactor Screenings (SIXFORS) were done in 0.5 L vessels(Sixfors multi-fermentation system, ATR Biotech, Laurel, Md.) under thefollowing conditions: pH at 6.5, 24° C., 0.3 SLPM, and an initialstirrer speed of 550 rpm with an initial working volume of 350 mL (330mL BMGY medium and 20 mL inoculum). IRIS multi-fermenter software (ATRBiotech, Laurel, Md.) was used to linearly increase the stirrer speedfrom 550 rpm to 1200 rpm over 10 hours, one hour after inoculation. Seedcultures (200 mL of BMGY in a 1 L baffled flask) were inoculateddirectly from agar plates. The seed flasks were incubated for 72 hoursat 24° C. to reach optical densities (OD₆₀₀) between 95 and 100. Thefermenters were inoculated with 200 mL stationary phase flask culturesthat were concentrated to 20 mL by centrifugation. The batch phase endedon completion of the initial charge glycerol (18-24 h) fermentation andwere followed by a second batch phase that was initiated by the additionof 17 mL of glycerol feed solution (50% [w/w] glycerol, 5 mg/L Biotin,12.5 mL/L PTM1 salts (65 g/L FeSO₄.7H₂O, 20 g/L ZnCl₂, 9 g/L H₂SO₄, 6g/L CuSO₄.5H₂O, 5 g/L H₂SO₄, 3 g/L MnSO₄.7H₂O, 500 mg/L CoCl₂.6H₂O, 200mg/L NaMoO₄.2H₂O, 200 mg/L biotin, 80 mg/L NaI, 20 mg/L H₃BO₄)). Uponcompletion of the second batch phase, as signaled by a spike indissolved oxygen, the induction phase was initiated by feeding amethanol feed solution (100% MeOH 5 mg/L biotin, 12.5 mL/L PTM1) at 0.6g/h for 32-40 hours. The cultivation is harvested by centrifugation.

Bioreactor cultivations (3 L) were done in 3 L (Applikon, Foster City,Calif.) and 15 L (Applikon, Foster City, Calif.) glass bioreactors and a40 L (Applikon, Foster City, Calif.) stainless steel, steam in placebioreactor. Seed cultures were prepared by inoculating BMGY mediadirectly with frozen stock vials at a 1% volumetric ratio. Seed flaskswere incubated at 24° C. for 48 hours to obtain an optical density(OD₆₀₀) of 20±5 to ensure that cells are growing exponentially upontransfer. The cultivation medium contained 40 g glycerol, 18.2 gsorbitol, 2.3 g K₂HPO₄, 11.9 g KH₂PO₄, 10 g yeast extract (BD, FranklinLakes, N.J.), 20 g peptone (BD, Franklin Lakes, N.J.), 4×10⁻³ g biotinand 13.4 g Yeast Nitrogen Base (BD, Franklin Lakes, N.J.) per liter. Thebioreactor was inoculated with a 10% volumetric ratio of seed to initialmedia. Cultivations were done in fed-batch mode under the followingconditions: temperature set at 24±0.5° C., pH controlled at to 6.5±0.1with NH₄OH, dissolved oxygen was maintained at 1.7±0.1 mg/L by cascadingagitation rate on the addition of O₂. The airflow rate was maintained at0.7 vvm. After depletion of the initial charge glycerol (40 g/L), a 50%glycerol solution containing 12.5 mL/L of PTM1 salts was fedexponentially at 50% of the maximum growth rate for eight hours until250 g/L of wet cell weight was reached. Induction was initiated after a30 minute starvation phase when methanol was fed exponentially tomaintain a specific growth rate of 0.01 h⁻¹. When an oxygen uptake rateof 150 mM/L/h was reached the methanol feed rate was kept constant toavoid oxygen limitation. The results are shown in Table 2, which showsabout a three-fold increase in antibody titer.

The antibodies were also analyzed to determine whether replacing thePichia pastoris PDI1 gene with an expression cassette encoding the humanPDI would have an effect on O-glycosylation of the antibodies. Ingeneral, O-glycosylation of antibodies intended for use in humans isundesirable.

O-glycan determination was performed using a Dionex-HPLC (HPAEC-PAD) asfollows. To measure O-glycosylation reduction, protein was purified fromthe growth medium using protein A chromatography (Li et al. Nat.Biotechnol. 24(2):210-5 (2006)) and the O-glycans released from andseparated from protein by alkaline elimination (beta-elimination)(Harvey, Mass Spectrometry Reviews 18: 349-451 (1999)). This processalso reduces the newly formed reducing terminus of the released O-glycan(either oligomannose or mannose) to mannitol. The mannitol group thusserves as a unique indicator of each O-glycan. 0.5 nmole or more ofprotein, contained within a volume of 100 μL PBS buffer, was requiredfor beta elimination. The sample was treated with 25 μL alkalineborohydride reagent and incubated at 50° C. for 16 hours. About 20 uLarabitol internal standard was added, followed by 10 μL glacial aceticacid. The sample was then centrifuged through a Millipore filtercontaining both SEPABEADS and AG 50W-X8 resin and washed with water. Thesamples, including wash, were transferred to plastic autosampler vialsand evaporated to dryness in a centrifugal evaporator. 150 μL 1%AcOH/MeOH was added to the samples and the samples evaporated to drynessin a centrifugal evaporator. This last step was repeated five moretimes. 200 μL of water was added and 100 μL of the sample was analyzedby high pH anion-exchange chromatography coupled with pulsedelectrochemical detection-Dionex HPLC (HPAEC-PAD). Average O-glycanoccupancy was determined based upon the amount of mannitol recovered.

As shown in Table 2, O-glycosylation was reduced in strains in which thePichia pastoris PDI1 was replaced with an expression cassette encodingthe human PDI. In strain yGLY733, O-glycan occupancy (number ofO-glycosylation sites O-glycosylated) was reduced and for those sitesoccupied, the percent of O-glycans consisting of only one mannose wasincreased. These results suggest that replacing the Pichia pastoris PDI1with an expression cassette encoding the human PDI will enable theproduction of antibodies in Pichia pastoris with reducedO-glycosylation.

TABLE 2 Anti-ADDL antibody: O-Glycan & Titer yGLY730 yGLY733 Pichia PDI1Wild-type Knockout Human PDI None Overexpressed O-glycan Occupancy(H2L2) 7.4 4.2 O-glycan % 75.5/24.5 82.5/17.5 (Man1/Man2) Titer 12.5mg/L (SixFors) 38.3 mg/L (SixFors) 93 mg/L (3L)

The above three strains (yGLY730, yGLY714, and yGLY733) produceglycoproteins that have Pichia pastoris N-glycosylation patterns. GS 2.0strains are Pichia pastoris strains that have been geneticallyengineered to produce glycoproteins having predominantly Man₅GlcNAc₂N-glycans. The following experiment was performed with GS 2.0 strainsthat produce glycoproteins that have predominantly Man₅GlcNAc₂ N-glycansto determine the effect of replacing the Pichia pastoris PDI1 proteinwith the human PDI protein on antibody titers produced by these strains.Strains yGLY2690 and yGLY2696 are GFI 2.0 strains that produceglycoproteins that have predominantly Man₅GlcNAc₂ N-glycans and have thePichia pastoris PDI1 gene replaced with the expression cassette encodingthe human PDI protein (See FIG. 3). These two strains were transformedwith plasmid vector pGLY2261, which encodes the anti-DKK1 antibody, toproduce strains yGLY3647 and yGLY3628 (See FIG. 3) and the strainsevaluated by 96 deep well screening as described above. Antibody wasproduced in 500 ml SixFors and 3 L fermentors using the parametersdescribed above to determine the effect of replacing the Pichia pastorisPDI1 protein with the human PDI protein on antibody titers produced bythe strains. The results are shown in Table 3. Strain yGLY2263 is acontrol in which plasmid vector pGLY2260 was transformed into strainyGLY645, which produces glycoproteins having predominantly Man5GlcNAc₂N-glycans and expresses only the endogenous PDI1 gene.

Table 3 shows that replacing the gene encoding the Pichia pastoris PDI1with an expression cassette encoding the human PDI in yeast geneticallyengineered to produce glycoproteins that have predominantly Man₅GlcNAc₂N-glycans effects an improvement in the titers of antibodies produced bythe yeast. Table 3 also shows that O-glycosylation occupancy was stillreduced in these strains genetically engineered to produce glycoproteinshaving predominantly Man₅GlcNAc₂ N-glycans. Additionally, Table 3 showsan increase in the amount of N-glycosylation in the strains with theendogenous PDI1 replaced with the human PDI.

TABLE 3 Anti-DKK1 antibody: Titer, N-glycan & O-glycan yGLY2263 GS2.0Strain (control) yGLY3647 yGLY3628 Pichia pastoris PDI1 Wild-typeKnockout Knockout Human PDI None Overexpressed Overexpressed Human ERO1αNone Expressed None Human GRP94 None None Expressed Pichia pastoris PRB1Intact Knockout Intact Pichia pastoris PEP4 Intact Intact KnockoutN-glycan (Man5) 83.7% 93.4% 95.4% O-glycan 23.7 9.2 10.0 (Occupancy:H2L2) O-glycan 55/40 88/12 87/13 (Man1/Man2) Titer 27 mg/L 61 mg/L 86mg/L (3L) (SixFors) (SixFors)

Example 2

A benefit of the strains shown in Tables 2 and 3 is that making yeaststrains that have replaced the endogenous PDI1 gene with an expressioncassette that encodes a heterologous PDI not only effects an increase inprotein yield but also effects a decrease in both the number of attachedO-glycans (occupancy) and a decrease in undesired Man₂ O-glycanstructures. Recombinant proteins produced in yeast often displayaberrant O-glycosylation structures relative to compositions of the sameglycoprotein produced from mammalian cell culture, reflecting thesignificant differences between the glycosylation machinery of mammalianand yeast cells. These aberrant structures may be immunogenic in humans.

The inventors noted that host cells of Pichia pastoris carrying thehuman PDI gene in place of the endogenous Pichia pastoris PDI1 gene werestrain more resistant to PMT protein inhibitors (See publishedInternational Application No. WO2007061631), suggesting that thesestrains might be better suited to tolerate deletions of various PMTgenes. This is because in prior attempts to make ΔPMT knockouts inΔOCH1/ΔPNO1/ΔPBS2 strains of Pichia pastoris, ΔPMT1 knockouts and ΔPMT2knockouts could not be obtained; presumably because they are lethal inthis genetic background (unpublished results). ΔPMT4 knockouts could beobtained, but they typically exhibited only weak growth and poor proteinexpression compared to parental strains (See FIGS. 6 and 7). While ΔPMT5and ΔPMT6 knockouts could be obtained, the deletions exhibited little orno effect on cell growth or protein expression compared to parentalstrains, suggesting that these PMT genes were not effective in reductionof O-glycosylation.

PMT knockout yeast strains were created in the appropriate Pichiapastoris strains following the procedure outlined for Saccharomycescerevisiae in Gentzsch and Tanner, EMBO J. 15: 25752-5759 (1996), asdescribed further in Published International Application No. WO2007061631. The nucleic acid molecules encoding the Pichia pastoris PMT1and PMT4 are shown in SEQ ID NOs: 47 and 49. The amino acid sequences ofthe Pichia pastoris PMT1 and PMT4 are shown in SEQ ID NOs: 48 and 50.The primers and DNA templates used for making the PMT deletions usingthe PCR overlap method are listed below.

To make a PMT1 knockout, the following procedure was followed. Three PCRreactions were set up. PCR reaction A comprised primers PMT1-KO1:5′-TGAACCCATCTGTAAATAGAATGC-3′ (SEQ ID NO: 17) and PMT1-KO2:5′-GTGTCACCTAAATCGTATGTGCCCATTTACTGGA AGCTGCTAACC-3′ (SEQ ID NO: 18) andPichia pastoris NRRL-Y11430 genomic DNA as the template. PCR reaction Bcomprised primers PMT1-KO3: 5′-CTCCCTATAGTGAGTCGTATTCATCATTGTACTTTGGTATATTGG-3′ (SEQ ID NO: 19) and PMT1-KO4:5′-TATTTGTACCTGCGTCCTGTTTGC-3′ (SEQ ID NO: 20) and Pichia pastorisNRRL-Y11430 genomic DNA as the template. PCR reaction C comprisedprimers PR29: 5′-CACATACGATTTAGGTGACAC-3′ (SEQ ID NO: 21) and PR32:5′-AATACGACTCACTATAGGGAG-3′ (SEQ ID NO: 22) and the template was plasmidvector pAG25 (Goldstein and McCusker, Yeast 15: 1541 (1999)). Theconditions for all three PCR reactions were one cycle of 98° C. for twominutes, 25 cycles of 98° C. for 10 seconds, 54° C. for 30 seconds, and72° C. for four minutes, and followed by one cycle of 72° C. for 10minutes.

Then in a second PCR reaction, primers PMT1-KO1+PMT1-KO4 from above weremixed with the PCR-generated fragments from PCR reactions A, B, and Cabove. The PCR conditions were one cycle of 98° C. for two minutes, 30cycles of 98° C. for 10 seconds, 56° C. for 10 seconds, and 72° C. forfour minutes, and followed by one cycle of 72° C. for 10 minutes.

The fragment generated in the second PCR reaction was gel-purified andused to transform appropriate strains in which the Pichia pastoris PDI1gene has been replaced with an expression cassette encoding the humanPDI1 protein. Selection of transformants was on rich media plates (YPD)containing 100 μg/mL nourseothricin.

To make a PMT4 knockout, the following procedure was followed. Three PCRreactions were set up. PCR reaction A comprised primers PMT4-KO1:5′-TGCTCTCCGCGTGCAATAGAAACT-3′ (SEQ ID NO: 23) and PMT4-KO2:5′-CTCCCTATAGTGAGTCGTATTCACAGTGTACCATCT TTCATCTCC-3′ (SEQ ID NO: 24) andPichia pastoris NRRL-Y11430 genomic DNA as the template. PCR reaction Bcomprised primers PMT4-KO3: 5′-GTGTCACCTAAATCGTATGTGAACCTAACTCTAATTCTTCAAAGC-3′ (SEQ ID NO: 25) and PMT4-KO4:5′-ACTAGGGTATATAATTCCCAAGGT-3′ (SEQ ID NO: 26) and Pichia pastorisNRRL-Y11430 genomic DNA as the template. PCR reaction C comprisedprimers PR29: 5′-CACATACGATTTAGGTGACAC-3′ (SEQ ID NO: 21) and PR32:5′-AATACGACTCACTATAGGGAG-3′ (SEQ ID NO: 22) and plasmid vector pAG25 asthe template.

The conditions for all three PCR reactions were one cycle of 98° C. fortwo minutes, 25 cycles of 98° C. for 10 seconds, 54° C. for 30 seconds,and 72° C. for four minutes, and followed by one cycle of 72° C. for 10minutes.

Then in a second PCR reaction, primers PMT4-KO1+PMT4-KO4 from above weremixed with the PCR-generated fragments from PCR reactions A, B, and Cabove. The PCR conditions were one cycle of 98° C. for two minutes, 30cycles of 98° C. for 10 seconds, 56° C. for 10 seconds, and 72° C. forfour minutes, and followed by one cycle of 72° C. for 10 minutes.

The fragment generated in the second PCR reaction was gel-purified andused to transform appropriate strains in which the Pichia pastoris PDI1gene has been replaced with an expression cassette encoding the humanPDI protein. Selection of transformants was on rich media plates (YPD)containing 100 μg/mL nourseothricin.

To test the ability of the strains to produce antibodies with reducedO-glycosylation, expression vectors encoding an anti-Her2 antibody andan anti-CD20 antibody were constructed.

Expression/integration plasmid vector pGLY2988 contains expressioncassettes encoding the heavy and light chains of an anti-Her2 antibody.Anti-Her2 heavy (HC) and light (LC) chains fused at the N-terminus toα-MAT pre signal peptide were synthesized by GeneArt AG. Each wassynthesized with unique 5′ EcoR1 and 3′ Fse1 sites. The nucleotide andamino acid sequences of the anti-Her2 HC are shown in SEQ ID Nos: 29 and30, respectively. The nucleotide and amino acid sequences of theanti-Her2 LC are shown in SEQ ID Nos: 31 and 32, respectively. Bothnucleic acid molecule fragments encoding the HC and LC fusion proteinswere separately subcloned using 5′ EcoR1 and 3′ Fse1 unique sites intoan expression plasmid vector pGLY2198 (contains the Pichia pastoris TRP2targeting nucleic acid molecule and the Zeocin-resistance marker) toform plasmid vector pGLY2987 and pGLY2338, respectively. The LCexpression cassette encoding the LC fusion protein under the control ofthe Pichia pastoris AOX1 promoter and Saccharomyces cerevisiae Cycterminator was removed from plasmid vector pGLY2338 by digesting withBamHI and NotI and then cloning the DNA fragment into plasmid vectorpGLY2987 digested with BamHI and NotI, thus generating the finalexpression plasmid vector pGLY2988 (FIG. 15).

Expression/integration plasmid vector pGLY3200 (map is identical topGLY2988 except LC and HC are anti-CD20 with α-amylase signalsequences). Anti-CD20 sequences were from GenMab sequence 2C6 exceptLight chain (LC) framework sequences matched those from VKappa 3germline. Heavy (HC) and Light (LC) variable sequences fused at theN-terminus to the α-amylase (from Aspergillus niger) signal peptide weresynthesized by GeneArt AG. Each was synthesized with unique 5′ EcoR1 and3′ KpnI sites which allowed for the direct cloning of variable regionsinto expression vectors containing the IgG1 and V kappa constantregions. The nucleotide and amino acid sequences of the anti-CD20 HC areshown in SEQ ID Nos: 37 and 38, respectively. The nucleotide and aminoacid sequences of the anti-CD20 LC are shown in SEQ ID Nos: 35 and 36,respectively. Both HC and LC fusion proteins were subcloned into IgG1plasmid vector pGLY3184 and V Kappa plasmid vector pGLY2600,respectively, (each plasmid vector contains the Pichia pastoris TRP2targeting nucleic acid molecule and Zeocin-resistance marker) to formplasmid vectors pGLY3192 and pGLY3196, respectively. The LC expressioncassette encoding the LC fusion protein under the control of the Pichiapastoris AOX1 promoter and Saccharomyces cerevisiae Cyc terminator wasremoved from plasmid vector pGLY3196 by digesting with BamHI and NotIand then cloning the DNA fragment into plasmid vector pGLY3192 digestedwith BamH1 and Not1, thus generating the final expression plasmid vectorpGLY3200 (FIG. 16).

Transformation of appropriate strains disclosed herein with the aboveanti-Her2 or anti-CD20 antibody expression/integration plasmid vectorswas performed essentially as follows. Appropriate Pichia pastorisstrains were grown in 50 mL YPD media (yeast extract (1%), peptone (2%),dextrose (2%)) overnight to an OD of between about 0.2 to 6. Afterincubation on ice for 30 minutes, cells were pelleted by centrifugationat 2500-3000 rpm for 5 minutes. Media was removed and the cells washedthree times with ice cold sterile 1M sorbitol before resuspension in 0.5ml ice cold sterile 1M sorbitol. Ten μL linearized DNA (5-20 ug) and 100μL cell suspension was combined in an electroporation cuvette andincubated for 5 minutes on ice. Electroporation was in a Bio-RadGenePulser Xcell following the preset Pichia pastoris protocol (2 kV, 25μF, 200Ω), immediately followed by the addition of 1 mL YPDS recoverymedia (YPD media plus 1 M sorbitol). The transformed cells were allowedto recover for four hours to overnight at room temperature (24° C.)before plating the cells on selective media.

Cell Growth conditions of the transformed strains for antibodyproduction was generally as follows. Protein expression for thetransformed yeast strains was carried out at in shake flasks at 24° C.with buffered glycerol-complex medium (BMGY) consisting of 1% yeastextract, 2% peptone, 100 mM potassium phosphate buffer pH 6.0, 1.34%yeast nitrogen base, 4×10⁻⁵% biotin, and 1% glycerol. The inductionmedium for protein expression was buffered methanol-complex medium(BMMY) consisting of 1% methanol instead of glycerol in BMGY. Pmtinhibitor (Pmti-3) in methanol was added to the growth medium to a finalconcentration of 0.2 μM, 2 μM, or 20 μM at the time the induction mediumwas added. Cells were harvested and centrifuged at 2,000 rpm for fiveminutes.

SixFors Fermenter Screening Protocol followed the parameters shown inTable 4.

TABLE 4 SixFors Fermenter Parameters Parameter Set-point ActuatedElement pH 6.5 ± 0.1 30% NH₄OH Temperature  24 ± 0.1 Cooling Water &Heating Blanket Dissolved O2 n/a Initial impeller speed of 550 rpm isramped to 1200 rpm over first 10 hr, then fixed at 1200 rpm forremainder of run

At time of about 18 hours post-inoculation, SixFors vessels containing350 mL media A (See Table 6 below) plus 4% glycerol were inoculated withstrain of interest. A small dose (0.3 mL of 0.2 mg/mL in 100% methanol)of Pmti-3(5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid) (See Published International Application No. WO 2007061631) wasadded with inoculum. At time about 20 hour, a bolus of 17 mL 50%glycerol solution (Glycerol Fed-Batch Feed, See Table 7 below) plus alarger dose (0.3 mL of 4 mg/mL) of Pmti-3 was added per vessel. At about26 hours, when the glycerol was consumed, as indicated by a positivespike in the dissolved oxygen (DO) concentration, a methanol feed (SeeTable 8 below) was initiated at 0.7 mL/hr continuously. At the sametime, another dose of Pmti-3 (0.3 mL of 4 mg/mL stock) was added pervessel. At time about 48 hours, another dose (0.3 mL of 4 mg/mL) ofPmti-3 was added per vessel. Cultures were harvested and processed attime about 60 hours post-inoculation.

TABLE 5 Composition of Media A Martone L-1 20 g/L Yeast Extract 10 g/LKH₂PO4 11.9 g/L K₂HPO₄ 2.3 g/L Sorbitol 18.2 g/L Glycerol 40 g/LAntifoam Sigma 204 8 drops/L 10X YNB w/Ammonium Sulfate w/o Amino Acids(134 100 mL/L g/L) 250X Biotin (0.4 g/L) 10 mL/L 500X Chloramphenicol(50 g/L) 2 mL/L 500X Kanamycin (50 g/L) 2 mL/L

TABLE 6 Glycerol Fed-Batch Feed Glycerol 50% m/m PTM1 Salts (see TableIV-E below) 12.5 mL/L 250X Biotin (0.4 g/L) 12.5 mL/L

TABLE 7 Methanol Feed Methanol 100% m/m PTM1 Salts 12.5 mL/L 250X Biotin(0.4 g/L) 12.5 mL/L

TABLE 8 PTM1 Salts CuSO4—5H2O 6 g/L NaI 80 mg/L MnSO4—7H2O 3 g/LNaMoO4—2H2O 200 mg/L H3BO3 20 mg/L CoCl2—6H2O 500 mg/L ZnCl2 20 g/LFeSO4—7H2O 65 g/L Biotin 200 mg/L H2SO4 (98%) 5 mL/L

O-glycan determination was performed using a Dionex-HPLC (HPAEC-PAD) asfollows. To measure O-glycosylation reduction, protein was purified fromthe growth medium using protein A chromatography (Li et al. Nat,Biotechnol. 24(2):210-5 (2006)) and the O-glycans released from andseparated from protein by alkaline elimination (beta-elimination)(Harvey, Mass Spectrometry Reviews 18: 349-451 (1999)). This processalso reduces the newly formed reducing terminus of the released O-glycan(either oligomannose or mannose) to mannitol. The mannitol group thusserves as a unique indicator of each O-glycan. 0.5 nmole or more ofprotein, contained within a volume of 100 μL PBS buffer, was requiredfor beta elimination. The sample was treated with 25 μL alkalineborohydride reagent and incubated at 50° C. for 16 hours. About 20 uLarabitol internal standard was added, followed by 10 μL glacial aceticacid. The sample was then centrifuged through a Millipore filtercontaining both SEPABEADS and AG 50W-X8 resin and washed with water. Thesamples, including wash, were transferred to plastic autosampler vialsand evaporated to dryness in a centrifugal evaporator. 150 μL 1%AcOH/MeOH was added to the samples and the samples evaporated to drynessin a centrifugal evaporator. This last step was repeated five moretimes. 200 μL of water was added and 100 of the sample was analyzed byhigh pH anion-exchange chromatography coupled with pulsedelectrochemical detection-Dionex HPLC (HPAEC-PAD). Average O-glycanoccupancy was determined based upon the amount of mannitol recovered.

FIGS. 4-7 show that the Pichia pastoris strains in which the endogenousPDI1 is replaced with a heterologous PDI from the same species as therecombinant protein to be produced in the strain and in which nativePMT1 or PMT4 genes have been deleted are capable of producingrecombinant human antibody at higher titers and with reducedO-glycosylation compared to production of the antibodies in strains thatcontain the endogenous PDI1 and do not have deletions of the PMT1 orPMT4 genes.

FIGS. 4A and 4B shows representative results from shakeflask (A) and 0.5L bioreactor (B) expression studies in which human anti-Her2 antibodywas produced in Pichia pastoris strains in which the human PDI gene(hPDI) replaced the endogenous PDI1 and strains in which the human PDIreplaced the endogenous PDI1 and the PMT1 gene disrupted (hPDI+Δpmt1).Antibodies were recovered and resolved by polyacrylamide gelelectrophoresis on non-reducing and reducing polyacrylamide gels. Undernon-reducing conditions, the antibodies remained intact whereas underreducing conditions, the antibodies were resolved into HCs and LCs.Lanes 1-2 shows antibodies produced from two clones produced fromtransformation of strain yGLY2696 with plasmid vector pGLY2988 encodingthe anti-Her2 antibody and lanes 3-6 shows the antibodies produced fromfour clones produced from transformation of strain yGLY2696 in which thePMT1 gene was deleted and with plasmid vector pGLY2988 encoding theanti-Her2 antibody. The Figures showed that the PMT1 deletion improvedantibody yield.

FIG. 5 shows representative results from a shakeflask expression studyin which human anti-DKK1 antibody was produced in Pichia pastorisstrains in which the human PDI gene (hPDI) replaced the endogenous PDI1and strains in which the human PDI replaced the endogenous PDI1 and thePMT1 gene is disrupted (hPDI+Δpmt1). Antibodies were recovered andresolved by polyacrylamide gel electrophoresis on non-reducing andreducing polyacrylamide gels. Under non-reducing conditions, theantibodies remained intact whereas under reducing conditions, theantibodies were resolved into HCs and LCs. Lanes 1 and 3 showsantibodies produced from two clones produced from transformation ofstrains yGLY2696 and yGLY2690 with plasmid vector pGLY2260 encoding theanti-DKK1 antibody and lanes 2 and 4 shows the antibodies produced fromtwo clones produced from transformation of strains yGLY2696 and yGLY2690in which the PMT1 gene was deleted with plasmid vector pGLY2260 encodingthe anti-DKK1 antibody. The figure shows that the PMT1 deletion improvedantibody yield.

FIG. 6 shows results from a 0.5 L bioreactor expression study wherehuman anti-Her2 antibody is produced in Pichia pastoris strains in whichthe human PDI replaced the endogenous PDI1 and the PMT4 gene isdisrupted (hPDI+Δpmt4), and strains that express only the endogenousPDI1 but in which the PMT4 gene is disrupted (PpPDI+Δpmt4). Antibodieswere recovered and resolved by polyacrylamide gel electrophoresis onnon-reducing polyacrylamide gels. Lanes 1 and 2 shows antibodiesproduced from two clones from transformation of strain yGLY24-1 withplasmid vector pGLY2988 encoding the anti-Her2 antibody and lanes 3-5show anti-Her2 antibodies produced from three clones produced fromtransformation of strain yGLY2690 in which the PMT4 gene was deleted.The figure shows that the PMT4 deletion improved antibody yield but inorder to have that improvement in yield, the cell must also have theendogenous PDI1 gene replaced with an expression cassette encoding thehuman PDI.

FIG. 7 shows results from a shakeflask expression study where humananti-CD20 antibody is produced in Pichia pastoris strains in which thehuman PDI replaced the endogenous PDI1 and the PMT4 gene disrupted(hPDI+Δpmt4) and strains that express only the endogenous PDI1 but inwhich the PMT4 gene is disrupted (PpPDI+Δpmt4). Antibodies wererecovered and resolved by polyacrylamide gel electrophoresis onnon-reducing and reducing polyacrylamide gels. Lane 1 shows antibodiesproduced from strain yGLY24-1 transformed with plasmid vector pGLY3200encoding the anti-CD20 antibody; lanes 2-7 show anti-CD20 antibodiesproduced from six clones produced from transformation of strain yGLY2690in which the PMT4 gene was deleted. The figure shows that the PMT4deletion improved antibody yield but in order to have that improvementin yield, the cell must also have the endogenous PDI1 gene replaced withan expression cassette encoding the human PDI.

Example 3

This example describes a chimeric BiP gene, in which the human ATPasedomain is replaced by the ATPase domain of Pichia pastoris KAR2, fusedto the human BiP peptide binding domain, under the control of the KAR2,or other ER-specific promoter from Pichia pastoris. The nucleotide andamino acid sequences of the human BiP are shown in Table 11 as SEQ IDNOs: 55 and 56, respectively. The nucleotide and amino acid sequences ofthe chimeric BiP are shown in Table 11 as SEQ ID NOs: 57 and 58,respectively. Further improvements in yield may be obtained by combiningthe replacement of the endogenous PDI1 gene, as described above, withthe use of chimeric BiP and human ERdj3 (SEQ D NOs: 76 and 77,respectively).

Example 4

This example demonstrates that occupancy of O-glycans in proteinsproduced in the above strains expressing the human PDI in place of thePichia pastoris PDI1 can be significantly reduced when either the Pichiapastoris Golgi Ca²⁺ ATPase (PpPMR1) or the Arabidopsis thaliana ER Ca²⁺ATPase (AtECA1) is overexpressed in the strains. In this example, theeffect is illustrated using glycoengineered Pichia pastoris strains thatproduce antibodies having predominantly Man5GlcNAc₂ N-glycans.

An expression cassette encoding the PpPMR1 gene was constructed asfollows. The open reading frame of P. pastoris Golgi Ca²⁺ ATPase(PpPMR1) was PCR amplified from P. pastoris NRRL-Y11430 genomic DNAusing the primers (PpPMR1/UP:5′-GAATTCATGACAGCTAATGAAAATCCTTTTGAGAATGAG-3′ (SEQ ID NO: 64) andPpPMR1/LP: 5′-GGCCGGCCTCAAACAGCCATGCTGTATCCATTGTATG-3′ (SEQ ID NO: 65).The PCR conditions were one cycle of 95° C. for two minutes; five cyclesof 95° C. for 10 seconds, 52° C. for 20 seconds, and 72° C. for 3minutes; 20 cycles of 95° C. for 10 seconds, 55° C. for 20 seconds, and72° C. for 3 minutes; followed by 1 cycle of 72° C. for 10 minutes. Theresulting PCR product was cloned into pCR2.1 and designated pGLY3811.PpPMR1 was removed from pGLY3811 by digesting with plasmid with PstI andFseI) and the PstI end had been made blunt with T4 DNA polymerase priorto digestion with FseI. The DNA fragment encoding the PpPMR1 was clonedinto pGFI30t digested with EcoRI with the ends made blunt with T4 DNApolymerse and FseI to generate pGLY3822 in which the PpPMR1 is operablylinked to the AOX1 promoter. Plasmid pGLY3822 targets the Pichiapastoris URA6 locus. Plasmid pGLY3822 is shown in FIG. 17. The DNAsequence of PpPMR1 is set forth in SEQ ID NO: 60 and the amino acidsequence of the PpPMR1 is shown in SEQ ID NO: 61.

An expression cassette encoding the Arabidopsis thaliana ER Ca²⁺ ATPase(AtECA1) was constructed as follows. A DNA encoding AtECA1 wassynthesized from GeneArt AG (Regensburg, Germany) and cloned to makepGLY3306. The synthesized AtECA1 was removed from pGLY3306 by digestingwith MlyI and FseI and cloning the DNA fragment encoding the AtECA1 intopGFI30t digested with EcoRI with the ends made blunt with T4 DNApolymerase and FseI to generate integration/expression plasmid pGLY3827.Plasmid pGLY3827 targets the Pichia pastoris URA6 locus. PlasmidpGLY3827 is shown in FIG. 18. The DNA sequence of the AtECA1 wascodon-optimized for expression in Pichia pastoris and is shown in SEQ IDNO: 62. The encoded AtECA1 has the amino acid sequence set forth in SEQID NO: 63.

Integration/expression plasmid pGLY3822 (contains expression cassetteencoding PpPMR1) or pGLY3827 (contains expression cassette encodingAtECA1) was linearized with SpeI and transformed into Pichia pastorisstrain yGLY3647 or yGLY3693 at the URA6 locus. The genomic integrationof pGLY3822 or pGLY3827 at URA6 locus was confirmed by colony PCR (cPCR)using primers, 5′AOX1 (5′-GCGACTGGTTCCAATTGACAAGCTT-3′ (SEQ ID NO: 66)and PpPMR1/cLP (5′-GGTTGCTCTCGTCGATACTCAAGTGGGAAG-3′ (SEQ ID NO: 67) forconfirming PpPMR1 integration into the URA6 locus, and 5′AOX1 andAtECA1/cLP (5′-GTCGGCTGGAACCTTATCACCAACTCTCAG-3′ (SEQ ID NO: 68) forconfirming integration of AtECA1 into the URA6 locus. The PCR conditionswere one cycle of 95° C. for 2 minutes, 25 cycles of 95° C. for 10seconds, 55° C. for 20 seconds, and 72° C. for one minute; followed byone cycle of 72° C. for 10 minutes.

Strain yGLY8238 was generated by transforming strain yGLY3647 withintegration/expression plasmid pGLY3833 encoding the PpPMR1 andtargeting the URA6 locus. In strain yGLY3647, the Pichia pastoris PDI1chaperone gene has been replaced with the human PDI gene as described inExample 1 and shown in FIGS. 3A and 3B.

Strain yGLY8240 was generated by transforming strain yGLY3647 withplasmid pGLY3827 encoding the AtECA1 and targeting the URA6 locus. Thegeneology of the strains is shown in FIGS. 3A and 3B.

The strains were evaluated for the effect the addition of PpPMR1 orAtECA1 to the humanized chaperone strains might have on reducingO-glycosylation of the antibodies produced by the strains. As shown inTable 9 the addition of either PpPMR1 or AtECA1 into strain yGLY3647effected a significant reduction in O-glycosylation occupancy comparedto strain yGLY3647 expressing the human PDI in place of the Pichiapastoris PDI1 or strain yGLY2263 expressing only the endogenous PDI1 butcapable of making antibodies with a Man₅GlcNAc₂ glycoform as strainyGLY3647. The results also suggest that yeast strains that express itsendogenous PDI1 and not the human PDI and overexpress a Ca²⁺ ATPase willproduce glycoproteins with reduced O-glycan occupancy.

TABLE 9 yGLY3647 + Ca²⁺ ATPase yGLY8240 yGLY8238 Strain yGLY2263yGLY3647 AtECA1 PpPMR1 O-glycan 23.7 9.2 5.54 6.28 occupancy (H2 + L2:anti- DKK1) O-glycan occupancy was determined by Mannitol assay.

Example 5

A DNA fragment encoding the human calreticulin (hCRT) without its nativesignal sequence was PCR amplified from a human liver cDNA library (BDBiosciences, San Jose, Calif.) using primers hCRT-BstZ17I-HA/UP:5′-GTATACCCATACGACGTCCCAGACTACGCTGAGCCCGCCGTCTACTTCAAGGAGC-3′ (SEQ IDNO: 73) and hCRT-PacI/LP:5′-TTAATTAACTACAGCTCGTCATGGGCCTGGCCGGGGACATCTTCC-3′ (SEQ ID NO: 74). ThePCR conditions were one cycle of 98° C. for two min; 30 cycles of 98° C.for 10 seconds, 55° C. for 30 seconds, and 72° C. for two minutes, andfollowed by one cycle of 72° C. for 10 minutes. The resulting PCRproduct was cloned into pCR2.1 Topo vector to make pGLY1224. The DNAencoding the hCRT further included modifications such that the encodedtruncated hCRT has an HA tag at its N-terminus and HDEL at itsC-terminus. The DNA encoding the hCRT was released from pGLY1224 bydigestion with BstZ17I and PacI and the DNA fragment cloned into anexpression vector pGLY579, which had been digested with NotI and PacI,along with a DNA fragment encoding the S. cerevisiae alpha-mating factorpre signal sequence having NotI and PacI compatible ends to createpGLY1230. This plasmid is an integration/expression plasmid that encodesthe hCRT with the S. cerevisiae alpha-mating factor pre signal sequenceand HA tag at the N-terminus and an HDEL sequence at its C-terminusoperably linked to the Pichia pastoris GAPDH promoter and targeting theHIS3 locus of Pichia pastoris.

A DNA fragment encoding the human ERp57 (hERp57) was synthesized byGeneArt AG having NotI and PacI compatible ends. The DNA fragment wasthen cloned into pGLY129 digested with NotI and PacI to producepGLY1231. This plasmid encodes the hERp57 operably linked to the Pichiapastoris PMA1 promoter.

Plasmid pGLY1231 was digested with SwaI and the DNA fragment encodingthe hERp57 was cloned into plasmid pGLY1230 digested with PmeI. Thus,integration/expression plasmid pGLY1234 encodes both the hCRT andhERp57. Plasmid pGLY1234 is shown in FIG. 19.

Strain yGLY3642 was generated by counterselecting strain yGLY2690 in thepresence of 5′FOA, a URA5 auxotroph.

Strain yGLY3668 was generated by transforming yGLY3642 withintegration/expression plasmid pGLY1234 encoding the hCRT and hERp57 andwhich targets the HIS3 locus.

Strain yGLY3693 was generated by transforming strain yGLY3668 withintegration/expression plasmid pGLY2261, which targets an expressioncassette encoding the anti-DKK1 antibody to the TRP2 locus.

Strain yGLY8239 was generated by transforming strain yGLY3693 withintegration/expression plasmid pGLY3833 encoding the PpPMR1 andtargeting the URA6 locus.

Strain yGLY8241 was generated by transforming strain yGLY3693 withintegration/expression plasmid pGLY3827 encoding the AtECA1 andtargeting the URA6 locus.

The geneology of the strains described in this example are shown inFIGS. 3A and 3B.

The above strains were evaluated to see whether the addition of hCRT andhERp57 to the humanized chaperone strains expressing PpPMR1 or AtECA1 ofthe previous example might effect a further reduction in O-glycanoccupancy of the antibodies produced. As shown in Table 10, in strainyGLY3693 expressing hCRT and hERp57 alone, there was about a 2-folddecrease in O-glycan occupancy, which was further decreased up to a4-fold in strains that further expressed PpPMR1 or AtECA1. The resultsalso suggest that yeast strains that express its endogenous PDI1 and notthe human PDI and overexpress a Ca²⁺ ATPase will produce glycoproteinswith reduced O-glycan occupancy.

TABLE 10 yGLY3693 + Ca²⁺ ATPase yGLY8241 yGLY8239 Strain yGLY2263yGLY3693 AtECA1 PpPMR1 O-glycan 23.7 10.43 5.59 7.86 occupancy (H2 + L2:anti- DKK1) O-glycan occupancy was determined by Mannitol assay.

TABLE 11 BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: DescriptionSequence 1 PCR primer AGCGCTGACGCCCCCGAGGAGGAGGACCAC hPDI/UP1 2PCR primer CCTTAATTAATTACAGTTCATCATGCACAGCTTTCTGATCAT hPDI/LP-PacI 3PCR primer ATGAATTCAGGC CATATCGGCCATTGTTTACTGTGCG PB248 CCCACAGTAG 4PCR primer ATGTTTA AACGTGAGGATTACTGGTGATGAAAGAC PB249 5 PCR primerAGACTAGTCTATTTGGAG ACATTGACGGATCCAC PB250 6 PCR primerATCTCGAGAGGCCATGCAGGCCAACCACAAGATGAATCAAAT PB251 TTTG 7 PCR primerGGTGAGGTTGAGGTCCCAAGTGACTATCAAGGTC PpPDI/UPi-1 8 PCR primerGACCTTGATAGTCACTTGGGACCTCAACCTCACC PpPDI/LPi-1 9 PCR primerCGCCAATGATGAGGATGCCTCTTCAAAGGTTGTG PpPDI/UPi-2 10 PCR primerCACAACCTTTGAAGAGGCATCCTCATCATTGGCG PpPDI/LPi-2 11 PCR primerGGCGATTGCATTCGCGAC TGTATC PpPDI-5′/UP 12 PCR primerCCTAGAGAGCGGTGG CCAAGATG hPDI-3′/LP 13 PCR primerGTGGCCACACCAGGGGGC ATGGAAC hPDI/UP 14 PCR primerCCTAGAGAGCGGTGG CCAAGATG hPDI-3′/LP 15 PCR primerAGCGCTGACGATGAAGTTGATGTGGATGGTACA GTAG hGRP94/UP1 16 PCR primerGGCCGGCCTTACAATTCATCATG TTCAGCTGTAGATTC hGRP94/LP1 17 PCR primerTGAACCCATCTGTAAATAGAATGC PMT1-KO1 18 PCR primerGTGTCACCTAAATCGTATGTGCCCATTTACTGGA PMT1-KO2 AGCTGCTAACC 19 PCR primerCTCCCTATAGTGAGTCGTATTCATCATTGTACTTT PMT1-KO3 GGTATATTGG 20 PCR primerTATTTGTACCTGCGTCCTGTTTGC PMT1-KO4 21 PCR primer CACATACGATTTAGGTGACACPR29 22 PCR primer AATACGACTCACTATAGGGAG PR32 23 PCR primerTGCTCTCCGCGTGCAATAGAAACT PMT4-KO1 24 PCR primerCTCCCTATAGTGAGTCGTATTCACAGTGTACCATCT PMT4-KO2 TTCATCTCC 25 PCR primerGTGTCACCTAAATCGTATGTGAACCTAACTCTAA PMT4-KO3 TTCTTCAAAGC 26 PCR primerACTAGGGTATATAATTCCCAAGGT PMT4-KO4 27 SaccharomycesATG AGA TTC CCA TCC ATC TTC ACT GCT GTT TTG TTC GCT cerevisiaeGCT TCT TCT GCT TTG GCT mating factor pre-signal peptide (DNA) 28Saccharomyces MRFPSIFTAVLFAASSALA cerevisiae mating factor pre-signalpeptide (protein) 29 Anti-Her2GAGGTTCAGTTGGTTGAATCTGGAGGAGGATTGGTTCAACCT Heavy chainGGTGGTTCTTTGAGATTGTCCTGTGCTGCTTCCGGTTTCAACA (VH + IgG1TCAAGGACACTTACATCCACTGGGTTAGACAAGCTCCAGGAA constantAGGGATTGGAGTGGGTTGCTAGAATCTACCCAACTAACGGTT region) (DNA)ACACAAGATACGCTGACTCCGTTAAGGGAAGATTCACTATCTCTGCTGACACTTCCAAGAACACTGCTTACTTGCAGATGAACTCCTTGAGAGCTGAGGATACTGCTGTTTACTACTGTTCCAGATGGGGTGGTGATGGTTTCTACGCTATGGACTACTGGGGTCAAGGAACTTTGGTTACTGTTTCCTCCGCTTCTACTAAGGGACCATCTGTTTTCCCATTGGCTCCATCTTCTAAGTCTACTTCCGGTGGTACTGCTGCTTTGGGATGTTTGGTTAAAGACTACTTCCCAGAGCCAGTTACTGTTTCTTGGAACTCCGGTGCTTTGACTTCTGGTGTTCACACTTTCCCAGCTGTTTTGCAATCTTCCGGTTTGTACTCTTTGTCCTCCGTTGTTACTGTTCCATCCTCTTCCTTGGGTACTCAGACTTACATCTGTAACGTTAACCACAAGCCATCCAACACTAAGGTTGACAAGAAGGTTGAGCCAAAGTCCTGTGACAAGACTCATACTTGTCCACCATGTCCAGCTCCAGAATTGTTGGGTGGTCCTTCCGTTTTTTTGTTCCCACCAAAGCCAAAGGACACTTTGATGATCTCCAGAACTCCAGAGGTTACATGTGTTGTTGTTGACGTTTCTCACGAGGACCCAGAGGTTAAGTTCAACTGGTACGTTGACGGTGTTGAAGTTCACAACGCTAAGACTAAGCCAAGAGAGGAGCAGTACAACTCCACTTACAGAGTTGTTTCCGTTTTGACTGTTTTGCACCAGGATTGGTTGAACGGAAAGGAGTACAAGTGTAAGGTTTCCAACAAGGCTTTGCCAGCTCCAATCGAAAAGACTATCTCCAAGGCTAAGGGTCAACCAAGAGAGCCACAGGTTTACACTTTGCCACCATCCAGAGATGAGTTGACTAAGAACCAGGTTTCCTTGACTTGTTTGGTTAAGGGATTCTACCCATCCGACATTGCTGTTGAATGGGAGTCTAACGGTCAACCAGAGAACAACTACAAGACTACTCCACCTGTTTTGGACTCTGACGGTTCCTTTTTCTTGTACTCCAAGTTGACTGTTGACAAGTCCAGATGGCAACAGGGTAACGTTTTCTCCTGTTCCGTTATGCATGAGGCTTTGCACAACCACTACACTCAAAA GTCCTTGTCTTTGTCCCCTGGTAAGTAA30 Anti-Her2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKG Heavy chainLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA (VH + IgG 1EDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFP constantLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP region)AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (protein)EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHN HYTQKSLSLSPGK 31 Anti-Her2 GACATCCAAATGACTCAATCCCCATCTTCTTTGTCTGCTTCCG lightTTGGTGACAGAGTTACTATCACTTGTAGAGCTTCCCAGGACGT chain (VL +TAATACTGCTGTTGCTTGGTATCAACAGAAGCCAGGAAAGGC KappaTCCAAAGTTGTTGATCTACTCCGCTTCCTTCTTGTACTCTGGTG constantTTCCATCCAGATTCTCTGGTTCCAGATCCGGTACTGACTTCAC region)TTTGACTATCTCCTCCTTGCAACCAGAAGATTTCGCTACTTAC (DNA)TACTGTCAGCAGCACTACACTACTCCACCAACTTTCGGACAGGGTACTAAGGTTGAGATCAAGAGAACTGTTGCTGCTCCATCCGTTTTCATTTTCCCACCATCCGACGAACAGTTGAAGTCTGGTACAGCTTCCGTTGTTTGTTTGTTGAACAACTTCTACCCAAGAGAGGCTAAGGTTCAGTGGAAGGTTGACAACGCTTTGCAATCCGGTAACTCCCAAGAATCCGTTACTGAGCAAGACTCTAAGGACTCCACTTACTCCTTGTCCTCCACTTTGACTTTGTCCAAGGCTGATTACGAGAAGCACAAGGTTTACGCTTGTGAGGTTACACATCAGGGTTTGTCCTCCCCAGTTACTAAGTCCTTCAACAGAGGAGAG TGTTAA 32 Anti-Her2 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP light KLLIYSASFLYchain (VL + SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQG Kappa TKVEIKRTVA APSVFIFPPSDEQLKSGTASVVC constantLNNFYPREAKVQWKVDNALQSGNSQESVTEQ region)DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 33 Alpha ATGGTTGCTT GGTGGTCCTT GTTCTTGTAC GGATTGCAAG amylaseTTGCTGCTCC AGCTTTGGCT signal peptide (from Aspergillus niger α- amylase)(DNA) 34 Alpha  WIVAWWSLFLY GLQVAAPALA amylase signal  peptide (fromAspergillus niger α- amylase) 35 Anti-CD20GAGATCGTTT TGACACAGTC CCCAGCTACT TTGTCTTTGT Light chainCCCCAGGTGA AAGAGCTACA TTGTCCTGTA GAGCTTCCCA VariableATCTGTTCC TCCTACTTGG CTTGGTATCA ACAAAAGCCA Region (DNA)GGACAGGCTC CAAGATTGTT GATCTACGAC GCTTCCAATAGAGCTACTGG TATCCCAGCT AGATTCTCTG GTTCTGGTTCCGGTACTGAC TTCACTTTGA CTATCTCTTC CTTGGAACCAGAGGACTTCT CTGTTTACTA CTGTCAGCAG AGATCCAATTGGCCATTGAC TTTCGGTGGT GGTACTAAGG TTGAGATCAAGCGTACGGTT GCTGCTCCTT CCGTTTTCAT TTTCCCACCATCCGACGAAC AATTGAAGTC TGGTACCCAA TTCGCCC 36 Anti-CD20EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP Light chainGQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP VariableEDFAVYYCQQ RSNWPLTFGG GTKVEIKRTV Region AAPSVFIFPPSDEQLKSGTQFA 37Anti-CD20 GCTGTTCAGC TGGTTGAATC TGGTGGTGGA TTGGTTCAAC Heavy chainCTGGTAGATC CTTGAGATTG TCCTGTGCTG CTTCCGGTTT VariableTACTTTCGGT GACTACACTA TGCACTGGGT TAGACAAGCT Region (DNA)CCAGGAAAGG GATTGGAATG GGTTTCCGGT ATTTCTTGGAACTCCGGTTC CATTGGTTAC GCTGATTCCG TTAAGGGAAGATTCACTATC TCCAGAGACA ACGCTAAGAA CTCCTTGTACTTGCAGATGA ACTCCTTGAG AGCTGAGGAT ACTGCTTTGTACTACTGTAC TAAGGACAAC CAATACGGTT CTGGTTCCACTTACGGATTG GGAGTTTGGG GACAGGGAAC TTTGGTTACTGTCTCGAGTG CTTCTACTAA GGGACCATCC GTTTTTCCATTGGCTCCATC CTCTAAGTCT ACTTCCGGTG GTACCCAATT CGCCC 38 Anti-CD20AVQLVESGGG LVQPGRSLRL SCAASGFTFG DYTMHWVRQA Heavy chainPGKGLEWVSG ISWNSGSIGY ADSVKGRFTI SRDNAKNSLY VariableLQMNSLRAED TALYYCTKDN QYGSGSTYGL GVWGQGTLVT RegionVSSASTKGPS VFPLAPSSKS TSGGTQFA 39 human PDIGACGCCCCCGAGGAGGAGGACCACGTCTTGGTGCTGCGGAAA Gene (DNA)AGCAACTTCGCGGAGGCGCTGGCGGCCCACAAGTACCCGCCGGTGGAGTTCCATGCCCCCTGGTGTGGCCACTGCAAGGCTCTGGCCCCTGAGTATGCCAAAGCCGCTGGGAAGCTGAAGGCAGAAGGTTCCGAGATCAGGTTGGCCAAGGTGGACGCCACGGAGGAGTCTGACCTAGCCCAGCAGTACGGCGTGCGCGGCTATCCCACCATCAAGTTCTTCAGGAATGGAGACACGGCTTTCCCCCAAGGAATATACAGCTGGCAGAGAGGCTGATGACATCGTGAACTGGCTGAAGAAGCGCACGGGCCCGGCTGCCACCACCCTGCCTGACGGCGCAGCTGCAGAGTCCTTGGTGGAGTCCAGCGAGGTGGCCGTCATCGGCTTCTTCAAGGACGTGGAGTCGGACTCTGCCAAGCAGTTTTTGCAGGCAGCAGAGGCCATCGATGACATACCATTTGGGATCACTTCCAACAGTGACGTGTTCTCCAAATACCAGCTCGACAAAGATGGGGTTGTCCTCTTTAAGAAGTTTGATGAAGGCCGGAACAACTTTGAAGGGGAGGTCACCAAGGAGAACCTGCTGGACTTTATCAAACACAACCAGCTGCCCCTTGTCATCGAGTTCACCGAGCAGACAGCCCCGAAGATTTTTGGAGGTGAAATCAAGACTCACATCCTGCTGTTCTTGCCCAAGAGTGTGTCTGACTATGACGGCAAACTGAGCAACTTCAAAACAGCAGCCGAGAGCTTCAAGGGCAAGATCCTGTTCATCTTCATCGACAGCGACCACACCGACAACCAGCGCATCCTCGAGTTCTTTGGCCTGAAGAAGGAAGAGTGCCCGGCCGTGCGCCTCATCACCTTGGAGGAGGAGATGACCAAGTACAAGCCCGAATCGGAGGAGCTGACGGCAGAGAGGATCACAGAGTTCTGCCACCGCTTCCTGGAGGGCAAAATCAAGCCCCACCTGATGAGCCAGGAGCTGCCGGAGGACTGGGACAAGCAGCCTGTCAAGGTGCTTGTTGGGAAGAACTTTGAAGACGTGGCTTTTGATGAGAAAAAAAACGTCTTTGTGGAGTTCTATGCCCCATGGTGTGGTCACTGCAAACAGTTGGCTCCCATTTGGGATAAACTGGGAGAGACGTACAAGGACCATGAGAACATCGTCATCGCCAAGATGGACTCGACTGCCAACGAGGTGGAGGCCGTCAAAGTGCACGGCTTCCCCACACTCGGGTTCTTTCCTGCCAGTGCCGACAGGACGGTCATTGATTACAACGGGGAACGCACGCTGGATGGTTTTAAGAAATTCCTAGAGAGCGGTGGCCAAGATGGGGCAGGGGATGTTGACGACCTCGAGGACCTCGAAGAAGCAGAGGAGCCAGACATGGAGGAAGACGATGACCAGAAAGCTGTGAAAGATGAACTGT AA 40 human PDIDAPEEEDHVLVLRKSNFAEALAAHKYPPVEFHAPWCGHCKALA Gene PEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKF (protein)FRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHGFPTLGFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDVDDLEDLEEAEEPDMEEDDDQKAVH DEL 41 Pichia ATGCAATTCAACTGGAATATTAAAACTGTGGCAAGTATTTTGT pastorisCCGCTCTCACACTAGCACAAGCAAGTGATCAGGAGGCTATTG PDI1 GeneCTCCAGAGGACTCTCATGTCGTCAAATTGACTGAAGCCACTTT (DNA)TGAGTCTTTCATCACCAGTAATCCTCACGTTTTGGCAGAGTTTTTTGCCCCTTGGTGTGGTCACTGTAAGAAGTTGGGCCCTGAACTTGTTTCTGCTGCCGAGATCTTAAAGGACAATGAGCAGGTTAAGATTGCTCAAATTGATTGTACGGAGGAGAAGGAATTATGTCAAGGCTACGAAATTAAGGGTATCCTACTTTGAAGGTGTTCCATGGTGAGGTTGAGGTCCCAAGTGACTATCAAGGTCAAAGACAGAGCCAAAGCATTGTCAGCTATATGCTAAAGCAGAGTTTACCCCCTGTCAGTGAAATCAATGCAACCAAAGATTTAGACGACACAATCGCCGAGGCAAAAGAGCCCGTGATTGTGCAAGTACTACCGGAAGATGCATCCAACTTGGAATCTAACACCACATTTTACGGAGTTGCCGGTACTCTCAGAGAGAAATTCACTTTTGTCTCCACTAAGTCTACTGATTATGCCAAAAAATACACTAGCGACTCGACTCCTGCCTATTTGCTTGTCAGACCTGGCGAGGAACCTAGTGTTTACTCTGGTGAGGAGTTAGATGAGACTCATTTGGTGCACTGGATTGATATTGAGTCCAAACCTCTATTTGGAGACATTGACGGATCCACCTTCAAATCATATGCTGAAGCTAACATCCCTTTAGCCTACTATTTCTATGAGAACGAAGAACAACGTGCTGCTGCTGCCGATATTATTAAACCTTTTGCTAAAGAGCAACGTGGCAAAATTAACTTTGTTGGCTTAGATGCCGTTAAATTCGGTAAGCATGCCAAGAACTTAAACATGGATGAAGAGAAACTCCCTCTATTTGTCATTCATGATTTGGTGAGCAACAAGAAGTTTGGAGTTCCTCAAGACCAAGAATTGACGAACAAAGATGTGACCGAGCTGATTGAGAAATTCATCGCAGGAGAGGCAGAACCAATTGTGAAATCAGAGCCAATTCCAGAAATTCAAGAAGAGAAAGTCTTCAAGCTAGTCGGAAAGGCCCACGATGAAGTTGTCTTCGATGAATCTAAAGATGTTCTAGTCAAGTACTACGCCCCTTGGTGTGGTCACTGTAAGAGAATGGCTCCTGCTTATGAGGAATTGGCTACTCTTTACGCCAATGATGAGGATGCCTCTTCAAAGGTTGTGATTGCAAAACTTGATCACACTTTGAACGATGTCGACAACGTTGATATTCAAGGTTATCCTACTTTGATCCTTTATCCAGCTGGTGATAAATCCAATCCTCAACTGTATGATGGATCTCGTGACCTAGAATCATTGGCTGAGTTTGTAAAGGAGAGAGGAACCCACAAAGTGGATGCCCTAGCACTCAGACCAGTCGAGGAAGAAAAGGAAGCTGAAGAAGAAGCTGAAA GTGAGGCAGACGCTCACGAGCTTTAA 42Pichia  MQFNWNIKTVASILSALTLAQASDQEAIAPEDSHVVKLTEATFES pastorisFITSNPHVLAEFFAPWCGHCKKLGPELVSAAEILKDNEQVKIAQI PDI1 GeneDCTEEKELCQGYEIKGYPTLKVFHGEVEVPSDYQGQRQSQSIVSY (protein)MLKQSLPPVSEINATKDLDDTIAEAKEPVIVQVLPEDASNLESNTTFYGVAGTLREKFTFVSTKSTDYAKKYTSDSTPAYLLVRPGEEPSVYSGEELDETHLVHWIDIESKPLEGDIDGSTFKSYAEANIPLAYYFYENEEQRAAAADIIKPFAKEQRGKINFVGLDAVKFGKHAKNLNMDEEKLPLEVIHDLVSNKKFGVPQDQELTNKDVTELIEKFIAGEAEPIVKSEPIPEIQEEKVFKLVGKAHDEVVEDESKDVLVKYYAPWCGHCKRMAPAYEELATLYANDEDASSKVVIAKLDHTLNDVDNVDIQGYPTLILYPAGDKSNPQLYDGSRDLESLAEFVKERGTHKVDAL ALRPVEEEKEAEEEAESEADAHDEL43 human EROlα GAAGAACAACCACCAGAGACTGCTGCTCAGAGATGCTTCTGT Gene (DNA)CAGGTTTCCGGTTACTTGGACGACTGTACTTGTGACGTTGAGACTATCGACAGATTCAACAACTACAGATTGTTCCCAAGATTGCAGAAGTTGTTGGAGTCCGACTACTTCAGATACTACAAGGTTAACTTGAAGAGACCATGTCCATTCTGGAACGACATTTCCCAGTGTGGTAGAAGAGACTGTGCTGTTAAGCCATGTCAATCCGACGAAGTTCCAGACGGTATTAAGTCCGCTTCCTACAAGTACTCTGAAGAGGCTAACAACTTGATCGAAGAGTGTGAGCAAGCTGAAAGATTGGGTGCTGTTGACGAATCTTTGTCCGAGAGACTCAGAAGGCTGTTTTGCAGTGGACTAAGCACGATGATTCCTCCGACAACTTCTGTGAAGCTGACGACATTCAATCTCCAGAGGCTGAGTACGTTGACTTGTTGTTGAACCCAGAGAGATACACTGGTTACAAGGGTCCAGACGCTTGGAAGATTTGGAACGTTATCTACGAAGAGAACTGTTTCAAGCCACAGACTATCAAGAGACCATTGAACCCATTGGCTTCCGGACAGGGAACTTCTGAAGAGAACACTTTCTACTCTTGGTTGGAGGGTTTGTGTGTTGAGAAGAGAGCTTTCTACAGATTGATCTCCGGATTGCACGCTTCTATCAACGTTCACTTGTCCGCTAGATACTTGTTGCAAGAGACTTGGTTGGAAAAGAAGTGGGGTCACAACATTACTGAGTTCCAGCAGAGATTCGACGGTATTTTGACTGAAGGTGAAGGTCCAAGAAGATTGAAGAACTTGTACTTTTTGTACTTGATCGAGTTGAGAGCTTTGTCCAAGGTTTTGCCATTCTTCGAGAGACCAGACTTCCCAATTGTTCACTGGTAACAAGATCCAGGACGAAGAGAACAAGATGTTGTTGTTGGAGATTTTGCACGAGATCAAGTCCTTTCCATTGCACTTCGACGAGAACTCATTTTTCGCTGGTGACAAGAAAGAAGCTCACAAGTTGAAAGAGGACTTCAGATTGCACTTCAGAAATATCTCCAGAATCATGGACTGTGTTGGTTGTTTCAAGTGTAGATTGTGGGGTAAGTTGCAGACTCAAGGATTGGGTACTGCTTTGAAGATTTTGTTCTCCGAGAAGTTGATCGCTAACATGCCTGAATCTGGTCCATCTTACGAGTTCCACTTGACTAGACAAGAGATCGTTTCCTTGTTCAACGCTTTCGGTAGAATCTCCACTTCCGTTAAAGAGTTGGAGAACTTCAGAAACTTG TTGCAGAACATCCACTAA 44human ER01α EEQPPETAAQRCFCQVSGYLDDCTCDVETIDRFNNYRLFPRLQKL Gene LESDYFRYYKVNLKRPCPFWNDISQCGRRDCAVKPCQSDEVPDG (protein)IKSASYKYSEEANNLIEECEQAERLGAVDESLSEETQKAVLQWTKHDDSSDNECEADDIQSPEAEYVDLLLNPERYTGYKGPDAWKIWNVIYEENCFKPQTIKRPLNPLASGQGTSEENTFYSWLEGLCVEKRAFYRLISGLHASINVHLSARYLLQETWLEKKWGHNITEFQQRFDGILTEGEGPRRLKNLYFLYLIELRALSKVLPFFERPDFQLFTGNKIQDEENKMLLLEILHEIKSFPLHFDENSFEAGDKKEAHKLKEDFRLHFRNISRIMDCVGCFKCRLWGKLQTQGLGTALKILFSEKLIANMPESGPSYEHLTRQEIVSLFNAFGRISTSVKELENFRNLLQNIH 45 human GRP94GATGATGAAGTTGACGTTGACGGTACTGTTGAAGAGGACTTG Gene (DNA)GGAAAGTCTAGAGAGGGTTCCAGAACTGACGACGAAGTTGTTCAGAGAGAGGAAGAGGCTATTCAGTTGGACGGATTGAACGCTTCCCAAATCAGAGAGTTGAGAGAGAAGTCCGAGAAGTTCGCTTTCCAAGCTGAGGTTAACAGAATGATGAAATTGATTATCAACTCCTTGTACAAGAACAAAGAGATTTTCTTGAGAGAGTTGATCTCTAACGCTTCTGACGCTTTGGACAAGATCAGATTGATCTCCTTGACTGACGAAAACGCTTTGTCCGGTAACGAAGAGTTGACTGTTAAGATCAAGTGTGACAAAGAGAAGAACTTGTTGCACGTTACTGACACTGGTGTTGGAATGACTAGAGAAGAGTTGGTTAAGACTTGGGTACTATCGCTAAGTCTGGTACTTCCGAGTTCTTGAACAAGATGACTGAGGCTCAAGAAGATGGTCAATCCACTTCCGAGTTGATTGGTCAGTTCGGTGTTGGTTTCTACTCCGCTTTCTTGGTTGCTGACAAGGTTATCGTTACTTCCAAGCACAACAACGACACTCAACACATTTGGGAATCCGATTCCAACGAGTTCTCCGTTATTGCTGACCCAAGAGGTAACACTTTGGGTAGAGGTACTACTATCACTTTGGTTTTGAAAGAAGAGGCTTCCGACTACTTGGAGTTGGACACTATCAAGAACTTGGTTAAGAAGTACTCCCAGTTCATCAACTTCCAATCTATGTTTGGTCCTCCAAGACTGAGACTGTTGAGGAACCAATGGAAGAAGAAGAGGCTGCTAAAGAAGAGAAAGAGGAATCTGACGACGAGGCTGCTGTTGAAGAAGAGGAAGAAGAAAAGAAGCCAAAGACTAAGAAGGTTGAAAAGACTGTTTGGGACTGGGAGCTTATGAACGACATCAAGCCAATTTGGCAGAGACCATCCAAAGAGGTTGAGGAGGACGAGTACAAGGCTTTCTACAAGTCCTTCTCCAAAGAATCCGATGACCCAATGGCTTACATCCACTTCACTGCTGAGGGTGAAGTTACTTTCAAGTCCATCTTGTTCGTTCCAACTTCTGCTCCAAGAGGATTGTTCGACGAGTACGGTTCTAAGAAGTCCGACTACATCAAACTTTATGTTAGAAGAGTTTTCATCACTGACGACTTCCACGATATGATGCCAAAGTACTTGAACTTCGTTAAGGGTGTTGTTGATTCCGATGACTTGCCATTGAACGTTTCCAGAGAGACTTTGCAGCAGCACAAGTTGTTGAAGGTTATCAGAAAGAAACTTGTTAGAAAGACTTTGGACATGATCAAGAAGATCGCTGACGACAAGTACAACGACACTTTCTGGAAAGAGTTCGGAACTAACATCAAGTTGGGTGTTATTGAGGACCACTCCAACAGAACTAGATTGGCTAAGTTGTTGAGATTCCAGTCCTCTCATCACCCAACTGACATCACTTCCTTGGACCAGTACGTTGAGAGAATGAAAGAGAAGCAGGACAAAATCTACTTCATGGCTGGTTCCTCTAGAAAAGAGGCTGAATCCTCCCCATTCGTTGAGAGATTGTTGAAGAAGGGTTACGAGGTTATCTACTTGACTGAGCCAGTTGACGAGTACTGTATCCAGGCTTTGCCAGAGTTTGACGGAAAGAGATTCCAGAACGTTGCTAAAGAGGGTGTTAAGTTCGACGAATCCGAAAAGACTAAAGAATCCAGAGAGGCTGTTGAGAAAGAGTTCGAGCCATTGTTGAACTGGATGAAGGACAAGGCTTTGAAGGACAAGATCGAGAAGGCTGTTGTTTCCCAGAGATTGACTGAATCCCCATGTGCTTTGGTTGGTTCCCAATACGGATGGAGTGGTAACATGGAAAGAATCATGAAGGCTCAGGCTTACCAAACTGGAAAGGACATCTCCACTAACTACTACGCTTCCCAGAAGAAAACTTTCGAGATCAACCCAAGACACCCATTGATCAGAGACATGTTGAGAAGAATCAAAGAGGACGAGGACGACAAGACTGTTTTGGATTTGGCTGTTGTTTTGTTCGAGACTGCTACTTTGAGATCCGGTTACTTGTTGCCAGACACTAAGGCTTACGGTGACAGAATCGAGAGAATGTTGAGATTGTCCTTGAACATTGACCCAGACGCTAAGGTTGAAGAAGAACCAGAAGAAGAGCCAGAGGAAACTGCTGAAGATACTACTGAGGACACTGAACAAGACGAGGACGAAAGAGATGGATGTTGGTACTGACGAAGAGGAAGAGACAGCAAAGGAATCCACTGCTGAACACGACGAGTTGTAA 46 human GRP94DDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQ Gene IRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDA (protein)LDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKIKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTEWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEEDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDE DEEMDVGTDEEEETAKESTAEHDEL47 PpPMT1 gene ACTTTTTCAATTCCTCAGGGTACTCCGTTGGAATTCTGTACTT (DNA)AGCAGCATACTGATCTTTGACCACCCAAGGAGCACCAGATCT CDS 3016-TTGCGATCTAGTCAACGTCAACTTGAGAAAAGTTTTCACGTAC 5385CACTTAGTGAACGCATTCCTATCACGGGAAACTTGATTTTCGTTCACGGTTACTTCTCCATCAGAGTTTGAGAGGCCAACGCGATAAGAGCAGTATCCTTCACGTACGGTACCATCAGGTAAGGTGATGGGAGCAAACCGTGCCTTTTCTCTGATGATCCCTTTATATCTGTTAGATCCAGCACTTTTAACATTCACTAGATCCCCAGGAAAAAATTCTTTCTTGAAGTGTAAATACACGTCATCGACTAATTGATCTAGTCTGGGTATGAGACTGAATTGCACATATCTCAAAATTGGTTCTCTGACAGGTTCTGGAAACTTATTTTCAACCGCTTGCATTTCCTCCTGTTCGTACTTGAGGGCCTCAAAGAAATCAAACGAGCTGTTTCCAGTGATTTCACACGCAAACTTCTTTTGGCTATAATAGTCCATCCTATCAAGGTACTCATCGTAGTTCAAAAACCACTCGCCAGTCTGTGGAATGTACCAAATTTCCGTGTCTAGATCATCAGGAAGCTGTTGTGGAGGGACAACTTCCACCTGCTTTCTTTTGAAGAGAACCATGGTGTTTGGGGATTAGAAGAAAACAAATATTTGAGCGGAACTTGCGAAAAAACGCCCCTAGCGAATGCAAGCTAGACATGTCAGGAAGATAAAATTGATACCGCAGAAGCAGGGGTAGTTGGGGAGGGCAATCAAGTACGTTCACAGAGCATGGCTGCGTTATCAACTGACTATTTTATGGCGTGGTTTAGAAGAGAGAGTATCAATTAGGCGTCAACTGGGACCATTATGATTAGACGTTGTAGGTAGATGCAGGTGAAAAATGGACAGACGTAGGCAACAAACACAAACTGTCGGGTAACCTTTAACAGTATTCAATTCCAGGTGTTTCAAGACAGCCTTAGATACTAGCAAGCTTCCAGGGAAACCCTATTACTCATGCTCCCACTGTTGGAACTCACAACCAAGAGGCTACATGTATGCGTATGCATACAGGTACTGCTCAGTGATAAATTTATTTCGCGAGATCGTACTCCAGAAACTTTCATGTAAGCCTTCCTACTTCGCTCTGCCCACTATGTTAGCCAGAAAGGTATTAGCTAGACAATGTCTGGTGGTAGCCAGGCTTTGTGCGGGTAGATTTGCCTCCTCATTATGCGGGTGCAGTTGTAGAGGTTTGATGAGGCCACCAAAATTTAACAGTTCCAAATCTCTTTCGAGATCGATGACCTCATCGTCCCTGTTTGAGTCTCCAAATTGTCCTTCCTGTGGTGTGGTTCTCCAAACAGAACATCCAGACAAAGATGGGTATTGTCTACTGCCCAAAGGTGAAAGGAAAGTTAAAAATTATCAAAATGAACTAAAGAAAGCTTTTTTTGAATGTGAAAAGGGAAGAACTTGCCGACAGACTGGGCCATGAGGTGGACTCTGAATCACTGATTATACCCAAGGAAATGTACCAAAAGCCCCGTACCCCGAAACGACTGGTTTGTCAGAGATGCTTCAAATCGCAAAACTATTCCTTGATCGACCATTCCATTCGTGAAGAAAATCCCGAACACAAGATCCTGGATGAGATCCCTTCAAACGCCAATATCGTCCACGTTTTATCTGCTGTTGATTTTCCTCTTGGTCTCAGCAAGGAACTGGTAAACAGATTTAAACCCACTCAGATTACGTACGTTATTACAAAGTCTGACGTGTTCTTCCCCGATAAGCTAGGTCTCCAACGGACGGGAGCTGCTTATTTTGAAGACAGCTTGGTAAAGCTTGTCGGTGCAGATCCTAGAAAGGTAGTATTGGTCTCAGGAAAAAGAAATTGGGGCCTCAAACAGCTGCTATCCACTTTACCCAGAGGTCCCAATTACTTTCTGGGAATGACGAACACCGGAAAATCAACCCTAATACGATCCATCGTTGGTAAGGATTACTCAAAGAAGCAGACAGAGAATGGCCCGGGTGTCTCTCACCTTCCTTCATTCACAAGAAAACCCATGAAGTTCAAAATGGACAACAACAGTCTTGAACTCGTAGATCTCCCTGGATACACTGCTCCAAATGGAGGTGTTTACAAGTATCTTAAGGAAGAGAACTACCGAGACATTTTGAACGTTAAACAGTTAAGCCATTGACATCCCTCAAGGCATACACAGAAACGTTGCCTTCGAAGCCAAAACTATTCAATGGTGTGCGAGTAATATGCATTGGTGGTTTAGTGTACATTCGGCCCCCAAAGGGTGTAGTGCTGAACAGTTTAGTCTCGTCAACCTTCCATCCTTCATGTACTCGTCGCTAAAAAAGGCCACCAGTGTAATCCAAGCGCCCCCACAAGCCTTGGTGAATTGCAGCGTCGTCAAGGAGGACAGTCCAGATGAACTGGTAAGATATGTGATCCCTCCATTTTATGGTTTAATTGACCTGGTCATTCAAGGTGTTGGATTTATCAAGCTTCTGCCCACTGGAGCTCGGAACACCAGAGAACTGATAGAAATTTTTGCCCCAAAAGACATCCAGCTCATGGTGCGTGATTCCATCCTCAAATACGTCTACAAGACCCATGCCGAACACGACTCAACCAATAATCTCCTGCATAAAAAGAACATAAAAGCCAGAGGCCAAACCATACTACGAAGACTACCCAAAAAGCCTGTATTCACAAAGCTTTTTCCCGTACCAGCCAACGTACCGTCTCATGAACTGCTCACCATGGTGACGGGAAAGGACGACCTAGCCGAGGAAGACAAAGAATACCGCTACGATATCCAGTATCCCAACAGATACTGGGATGAAACCATCTGTAAATAGAATGCTTATGTAATCAAGCACTTTCTGAAATTC

TGCCAGATATTTCTCCCGCAAAACGTAACACGTTGTTCTGTTTCCCTTTTGACAATGAGTAAAACAAGTCCTCAAGAGGTGCCAGAAAACACTACTGAGCTTAAAATCTCAAAAGGAGAGCTCCGTCCTTTTATTGTGACCTCTCCATCTCCTCAATTGAGCAAGTCTCGTTCTGTGACTTCAACCAAGGAGAAGCTGATATTGGCTAGTTTGTTCATATTTGCAATGGTCATCAGGTTCCACAACGTCGCCCACCCTGACAGCGTTGTGTTTGATGAAGTTCACTTTGGGGGGTTTGCCAGAAAGTACATTTTGGGAACCTTTTTCATGGATGTTCATCCGCCATTGGCCAAGCTATTATTTGCTGGTGTTGGCAGTCTTGGTGGATACGATGGAGAGTTTGAGTTCAAGAAAATTGGTGACGAATTCCCAGAGAATGTTCCTTATGTGCTCATGAGATATCTTCCCTCTGGTATGGGAGTTGGAACATGTATTATGTTGTATTTGACTCTGAGAGCTTCTGGTTGTCAACCAATAGTCTGTGCTCTGACAACCGCTCTTTTGATCATTGAGAATGCTAATGTTACAATCTCCAGATTCATTTTGCTGGATTCGCCAATGCTGTTTTTTATTGCTTCAACAGTTTACTCTTTCAAGAAATTTCAAATTCAGGAACCGTTTACCTTCCAATGGTACAAGACCCTTATTGCTACTGGTGTTTCTTTAGGGTTAGCAGCTTCCAGTAAATGGGTTGGTTTGTTCACCGTTGCCTGGATTGGATTGATAACAATTTGGGACTTATGGTTCATCATTGGTGATTTGACTGTTTCTGTAAAGAAAATTTTCGGCCATTTTATCACCAGAGCTGTAGCTTTCTTAGTCGTCCCCACTCTGATCTACCTCACTTTCTTTGCCATCCATTTGCAAGTCTTAACCAAGGAAGGTGATGGTGGTGCTTTCATGTCTTCGTCTTCAGATCGACCTTAGAAGGTAATGCTGTTCCAAAACAGTCGCTGGCCAACGTTGGTTTGGGCTCTTTAGTCACTATCCGTCATTTGAACACCAGAGGTGGTTACTTACACTCTCACAATCATCTTTACGAGGGTGGTTCTGGTCAACAGCAGGTCACCTTGTACCCACACATTGATTCTAATAATCAATGGATTGTACAGGATTACAACGCGACTGAGGAGCCAACTGAATTTGTTCCATTGAAAGACGGTGTCAAAATCAGATTAAACCACAAATTGACTTCCCGAAGATTGCACTCTCATAACCTCAGACCTCCTGTGACTGAACAAGATTGGCAAAATGAGGTATCTGCTTATGGACATGAGGGCTTTGGCGGTGATGCCAATGATGACTTTGTTGTGGAGATTGCCAAGGATCTTTCAACTACTGAAGAAGCTAAGGAAAACGTTAGGGCCATTCAAACTGTTTTTAGATTGAGACATGCGATGACTGGTTGTTACTTGTTCTCCCACGAAGTCAAGCTTCCCAAGTGGGCATATGAGCAACAAGAGGTTACTTGTGCTACTCAAGGTATCAAACCACTATCTTACTGGTACGTTGAGACCAACGAAAACCCATTCTTGGATAAAGAGGTTGATGAAATAGTTAGCTATCCTGTTCCGACTTTCTTTCAAAAGGTTGCCGAGCTACACGCCAGAATGTGGAAGATCAACAAGGGCTTAACTGATCATCATGTCTATGAATCCAGTCCAGATTCTTGGCCCTTCCTGCTCAGAGGTATAAGCTACTGGTCAAAAAATCACTCACAAATTTATTTCATAGGTAATGCTGTCACTTGGTGGACAGTCACCGCAAGTATTGCTTTGTTCTCTGTCTTTTTGGTTTTCTCTATTCTGAGATGGCAAAGAGGTTTTGGGTTCAGCGTTGACCCAACTGTGTTCAACTTCAATGTTCAAATGCTTCATTACATCCTAGGATGGGTACTGCATTACTTGCCATCTTTCCTTATGGCCCGTCAGCTATTTTTGCACCACTATCTACCATCATTGTACTTTGGTATATTGGCTCTCGGACATGTGTTTGAGATTATTCACTCTTATGTCTTCAAAAACAAACAGGTTGTGTCTTACTCCATATTCGTTCTCTTTTTTGCCGTTGCGCTTTCTTTCTTCCAAAGATATTCTCCATTGATCTATGCAGGACGATGGACCAAGGACCAATGCAACGAATCCAAGATACTCAAGTGGGACTTTGACTGTAACACCTTCCCCAGTCACACATCTCAGTATGAAAATATGGGCATCCCCTGTACAAACTTCCACTCCTAAAGAAGGAACCCACTCAGAATCTACCGTCGGAGAACCTGACGTTGAGAAGCTGG

ATCTATGACACAAGTTTATGGTTATTTGTCTTATGTAAGCAATATTTGGATTGATGTCTCGAGACCATCAACTCCATCACTGATAAGTTGATCGGATTTGTATTTCTGTCCCCTATTTACTAATTCCCTTTCCAGAAATAGATCATGAATGAGGCAGAATATAAGTGCCAAAGATGCCGGCTGCCGTTGACCATAGACGGATCTCTGGAAGACCTTAGCATATCACAGGCCAATCTTTTGACGGGACGAAATGGGAACTTTACAAAGAACACAATCCCCTTGGAGGATGCCGTGGAAGAAGATTTACCCAAGGTGCCTCAGAGCCGACTTAACCTCTTTAAAGAGGTCTACCAGAAGATGGATCACGATTTTACCAATGCCAGAGATGAATTTGTTGTGTTGAACAAGCACAATGATAACAGCGACGTCAATGTGGAGTATGATTACGAAGAAAACAACACTATCAGTCGTAGAATCAACACAATGACGAATATCTTCAATATCCTCAGCAACAAGTACGAAATTGATTTTCCGGTTTGCTACGAATGCGCCACATTGCTGATGGAGGAATTGAAGAATGAGTACGAAAGGGTCAATGCTGATAAAGAAGTTTACGCAAAGTTTCTATCCAAGCTTCGCAAACAGGACGCAGGTACAAATATGAAAGAAAGAACTGCTCAACTACTGGAGCAATTGGAGAAAACTAAGCAAGAAGAGAGAGATAAAGAAAAGAAGCTCCAAGGCCTATATGATGAAAGAGATAGTTTGGAAAAGGTATTAGCTTCTTTAGAGAATGAAATGGAACAGTTGAATATTGAAGAGCAGCAAATTTTTGAATTAGAGAACAAATATGAATATGAGTTAATGGAGTTCAAGAATGAGCAAAGCAGAATGGAAGCAATGTATGAGGATGGTTTGACGCAATTAGATAATTTAAGAAAAGTGAACGTCTTTAATGACGCTTTCAATATCTCGCATGATGGTCAATTCGGCACTATAAATGGGCTCAGGTTGGGCACGTTAGACAGTAAGAGGGTTTCTTGGTATGAATAAATGCTGCGTTGGGTCAAGTTGTTTTGTTACTCTTCACGTTATTGAGCAGACTTGAGCTTGAGCTCAAACATTACAAGATTTTTCCCATTGGCTCGACTTCCAAGATTGAATACCAAGTTGACCCAGATTCCAAACCTGTTACTATTAACTGCTTTTCTTCGGGAGAACAGTTACTGGATAAGCTTTTTCATTCTAATAAACTAGATCCTGCTATGAACGCAATCCTAGAAATCACTATTCAAATTGCAGATCATTTCACAAAACAAGATCCAACAAACGAATTGCCCTACAAAATGGAGAACGAAACAATATCAAACTTGAATATCAAACCTTCCAAACGTAAATCCAACGAGGAATGGACTTTGGCATGCAAACATCTGTTGACCAATCTCAAATGGATAATTGCCTTCAGTAGTTCAACGTGAACTAGTGTATTTAAAAAAAAGAAACAGAAACTTTATTGGATTATAAAACTATTTATCAAGTTCAAATTAACATAGCGACGAAGAGACCAGCTGCGGCTAAGACTGAACTACCTAGTACCGCTTGGGCACCGTTACCAGTTTCTGTACCTGTGCCAGTGGTACCAGTACCAGTACCGGTTCCAGTGCCAGTTCCTGTGCCTGTGCCTGTGCCTGTGCCGGTTCCGGTTCCAGTGCCTGTGCCTGTGCCCGTGCCTGTTGCAGTGGTATTAGTGAAACCTCCTGTGCCAGTTGCAGTGGTATTAGTAAATCCTCCTGTTCCTGTGGTGTTTGTGAGTCCTCCAGTTTCGGTCAAGTTTCCAGGAACACTAACATCAGGGGTTGAAGTGATCTCTGGTGGCACCGTGGGGACTGTGACATTGACATCATTTGTGAAGATTGGCTCCAACTCAGTTGTAGCCTTAACAACGCTTAATGCGAGAGTTGCACCGATCAAACTTTTGAATTGCATTTTACTTTTGTTACTTCTAAAATGAGATGAGGAAAGAAAGAAGAGAGAAGTGGAAGCACTGAAAGTGTGGTGTTATATCTGAAAAATTCATTACCAATCAAAACGTCAGACGATGATATGTCTAAGCCCGTGCAGAAACGTCTAGATCTTTTCAAACGTAAAGTACTTCCCCTTTTGGCACATCGTGGACTTGCTATTCCAAATATAGACGGGGACCTTTTTTAGAGTATCCCCGGGCGCCTCGAATTCTGGGGTATTTTTTTGCTATAGCATGAATTGGCAATAGGGATTGGGGACAACGTGTTTGACAGAAGACGTGTGTGTCCTGCCAAAAAGGGGTAAAGGTGCATTTGCCAAGGCCTGTGAATGATCTGAACACTAGAGGAAAGCAAGAAGGCTGTGTCGTAGTCTGTATTGGCTGTGTTGTCGCTGTGTCGGTTGCTTCAAAACTTTATTCGAGTCCGGTACGCGTCAATGGGTATTTTTCAAAAAGTTTCTAACTCCCTCAATCAACTTTGGTTTTGGCCGGATATGGCATGCCAGAAAAGGAAGTTTTACTCCTGGCGATGATGTTTACAAATCAAGCTTAGAGGGAGTAACCAATGCAGATAAGTTTGCGATGGCGCTGATCTTTATGCTCTCAACACCTTCTCGACTATTCAGGGTCATTTCGTGGCTTTGTATTTCGGGCACAACTGATCACCGAGGATCAATGAAATTTTCATGCACATCACTGATCCAGTTTCTGTCGAATTTGCAATTCCAGTTGATTGCAGGACCCGCGTTCTGCCTACACATTTTCTCGTGATTGTGGAAGTAATTCTAATTGACAGTCGATCACCACAATGACAATCTTAGTTGACCTTAGATTCCAGTGGAATGCAGTTGAATTGTCTTTTCGTTTAATTAGAGGAGAGTAACGGACCAGGGGCTCCTTTATTGTATATAATAATTATAATTTTTTTCACTATTTCACCTTTTCGCTTGGAATATAAAATTCTAATTATAATTCAACAGGAAATATTGTCCAA ACCACATGAAGTTGTCATG 48PpPMT1 gene MCQIFLPQNVTRCSVSLLTMSKTSPQEVPENTTELKISKGELRPFI (protein)VTSPSPQLSKSRSVTSTKEKLILASLFIFAMVIRFHNVAHPDSVVFDEVHFGGFARKYILGTFFMDVHPPLAKLLFAGVGSLGGYDGEFEFKKIGDEFPENVPYVLMRYLPSGMGVGTCIMLYLTLRASGCQPIVCALTTALLIIENANVTISRFILLDSPMLFFIASTVYSFKKFQIQEPFTFQWYKTLIATGVSLGLAASSKWVGLFTVAWIGLITIWDLWFIIGDLTVSVKKIFGHFITRAVAFLVVPTLIYLTFFAIHLQVLTKEGDGGAFMSSVFRSTLEGNAVPKQSLANVGLGSLVTIRHLNTRGGYLHSHNHLYEGGSGQQQVTLYPHIDSNNQWIVQDYNATEEPTEFVPLKDGVKIRLNHKLTSRRLHSHNLRPPVTEQDWQNEVSAYGHEGFGGDANDDFVVEIAKDLSTTEEAKENVRAIQTVFRLRHAMTGCYLFSHEVKLPKWAYEQQEVTCATQGIKPLSYWYVETNENPFLDKEVDEIVSYPVPTFFQKVAELHARMWKINKGLTDHHVYESSPDSWPFLLRGISYWSKNHSQIYFIGNAVTWWTVTASIALFSVFLVFSILRWQRGFGFSVDPTVFNFNVQMLHYILGWVLHYLPSFLMARQLFLHHYLPSLYFGILALGHVFEIIHSYVEKNKQVVSYSIFVLFFAVALSFFQRYSPLIYAGRWTKDQCNESKILKWDFDCNTFPSHTSQYEIWASPV QTSTPKEGTHSESTVGEPDVEKLGETV49 PpPMT4 TAGTAAAGAAATCTTGCAGTTTAATTCTTCCTCTTGTGTTTTTA (DNA)GCGATGAGACATCGGCACTCAGAGTTAAGTTTGCTTGCATCTG CDS 3168-CTCTGATAACTTTTGCTGTGACTCTGTTGCAATGCTTTTGGTA 5394ACGGTCAATTCGTCTATGGTTTGTTGATACTTTGACTTTAAGGCAGTAATATTGTCCTGTAGTTTATCATTATATGCTTCCAATGTTTTGACCTTTGATGAAATGTTTTTTCGATTAACAGTTAGTTCATCGAAGGAGAGCTCCAACTCTGATACTTGCATTCTTAAATTATTTATAATGGTATCCTTAACTTCTAGTGATTTCGAGTGGCTTGCCTGGGCACTCTTAAGTTCTTTTCTCAACTGTGCTATGGATGGCTCAAGCACTAGAATTTGTTTCTCTGAATCGAATAATTTTATTTCTAGCTTCTGAGCAAGCTCACAGGCGCTTACTTTTTCGGAAGTTAGAAACTTTGCTTCGTTATTCATGGCAGACAGTTCTATTCTTAATTGCTTATTTTCTTTCCTAACTTCCAAAATCTCCGATTCCAGGGGTTCATATCTACGGGAGGAAACCTGATTGCATGACTTTTCGAACGTTTTTTGATCAGAAAGTTGACAGATTGTGCCATCAGTTGACGAGACAGCCTCAAACTGAGTTGCTTCCATGTTGCACAAATTATCATTGAATTCAGCCACTTCTTTCTCCAAATCTCCGTTTACCAGCTCCTTCTTCTTTCCTGAAGCAATAGATGATGATCGATGAATATAGTCCTCTTTCAATGGGTTTTGGATCTCTTTGTCCCATTGACAAGAAGCTATGCTCCTTGAATCCTTCATTGACATTGGGTATGAAATTTTGCTACCATCTACCTTTGCACTAATTTCTGTGGGCGAATTGTGTGTTTTCAGTAGATCTTCAAGTGCTTTCTTTTCGTTTTCTATCTTCATAAGAGATATTCTCAATTTATTTACGGTGTCAGTGGCAGACAAATTGTTTAATGAAACGGTATCCAGAGACTCCTGCTCCAGGTACTCTGACTGAGCTACAGGGAAGGGTTTCTTGTCGTGTATGGAGAACTTCTGCTCAAGTTGGGCTTTCAAGGAATTCACTTGGTGATGCAAAAGCTCATTTTCCTCATTCAAAGAAGTATTCATATTTTTAAGTTCCTCCAGCTCAAACGTACTGCGGCCTTCCAAAGTGCAGATTTTATCCTTAAGACTCAATTTCTCATTCTTCAAACTTTCCATCTCTCTATTGAGCGTTTCAACCTGGTCAGTTTTCAACTTGAGTTCTTCTGAAAATTTGATACTTGAACTTTTAGCAAGGGAAGCTTCATCAAAAGTATCTTGTAGCTTGGTTTCTAAAGACCAGTTAGAATCAAGTAGCCTTTGCTGCTCTTGTTCCAACTTTTTGGAAAAAATTGCCTGGTGAGTGATCTTTTCAGCGAGATCCTCATTTTCTTTAACTTTTTGCTTCAATAGAGATTTGAGTTTTTGATTATCAGAGTTCAGCTTCCGACATTCGACTAAAAGGTTGTCACTAATACCTGTTAAAAAATCTATTTTGTTCGTCTTTTTTTTGCTTGGCGACTTTAGAGGTAATGCAGGAAGGGAAGGATGAAATTCTGACTCCTGGTGCCGATTTCTCAGCTTTCTCGGAAGTGGGGGTAGCTGAAATGGTACATTGTGGTTCGTATCACCAAGATCCATTTTTATGTCTTTGTCCATTAGAAAATTCAAGAATCTTTCAAAAAAAATAGAAACAGAAGATTTAGTAAACTTAGGTGAGGTGATATAAACCTAATTGCCTGTTTTATTTTGATCATGTATGTAAATTGTGAAAGGTAAATACGCGAAACTTATGTATGTATTTGCAAAGATGCACAAGACACACAAGGATTAATGGGCTATTTGCTCTACATTCGCAAAAAATAGCCAGCATTTATTTTTTGAATGGATACTCAATAAGCCCATCCCTACGCTTCCATATCTTTTTTTTCTTTTTGGTAGTAACATGCTCCACGAATACCTCTTCACAAGTAGATTTTTTAAATGAGCGGATAAAGCGGGGGTCCCATAGTTCACTAGCAACTCCTAAGTCTTTGCAGCATCTCATTAAAGCATTGCTCTTACAGCCTTCAGTAGCAGTAGGAATTCCCTTCTCTGAAAAAAAATCTTGCTCTCCGCGTGCAATAGAAACTAGTCGGCCCTGTACAATTAAAGCATACTCCCTGGTTAAAGTACCTCCTCCGAACTTGCTCTTGTTGATCAAAGTTTCTGACCTTGGGGCCAGTCCCCAGCCACCAGGGCCAAACGCTTTATTGAGGATACGACGATACTTAATCTCTGGAAGATAAGTAGTCCATCTGGTGTGATTTCGACATCTTCGTTGCTAATTGGTTGACATAATATGTTACTACTTTCATTACTGAAGGAGCAAATACCTAGTCCATGGAACGAATCCGACCAATTGATTCCATCGCCACTTGTATTAGAGATTGGGGTGTCGTTTAACTGTGAAGTTCCAAACAAAATTGATAAACTGCTCTCGTTCTTAGCTTGGCCACTTTTGGAGTCTCAATAGTAGCGTTTTGGCTCTCGTGAATTTTCGTCACAGAGTCGGATGAAGAAGGTGCAAATGCTTCTAGCATTGTAGAGTCGACCACATAGAACCTTTTTAAAGAGTTATGAAAATAACTCTTGGTAGGGCCAAATACAACCCGATATCGTCTTAGCATAAGAGCTGCTTCTTTGGAATATCGTTTCTTGTAAGTAATTACGTGTTGGCTAAACACTTAGAAGTCAGTCGCGCATGCGGCCAAAAACAGATAGGGATAGAAGATGAACTGACAAAAACATCAAGAAGGTGAAGACATTCATTCTATGAAAACTAGTTTTTATATAAAATTATGGTCTGCATTTAGAGAGCAATGATGTAATCAAACATCAATAAGTGCTTGTCGCATCAATATTTAATAGGTAATCATGGAGTATTCTAGTCTACCGCCTTAAAAAAAGCTCACTCGATCTAGTGCAGCTTGATTGTGTACTTCAATAGTATTCCAACGACCTTAACATCTTAACACCATGTAAATTTAAGATCCACGTATACGAT

TAAAATCAAGAAAGAGATCGAGAAAAGTTTCTTTGAACACTGAAAAGGAGCTGAAAAATAGCCATATTTCTCTTGGAGATGAAAGATGGTACACTGTGGGTCTTCTCTTGGTGACAATCACAGCTTTCTGTACTCGATTCTATGCTATCAACTATCCAGATGAGGTTGTTTTTGACGAAGTTCATTTCGGAAAATTTGCTAGCTACTATCTAGAGCGTACTTATTTTTTTGATCTGCACCCTCCGTTTGCCAAGCTCCTGATTGCGTTTGTCGGCTTTTTAGCTGGGTACAATGGTGAGTTCAAGTTTACAACTATTGGTGAATCTTATATCAAAAACGAGGTTCCCTACGTAGTTTACAGATCATTGAGCGCTGTGCAAAGGATCTTTAACGGTGCCAATTGTTTATTTGTGTCTCAAAGAATGCGGATATACAGTTTTGACTTGTGTTTTTGGTGCATGTATCATATTGTTTGATGGGGCCCACGTTGCTGAGACTAGACTAATCTTGCTGGATGCCACGTTGATTTTTTTCGTTTCATTGTCCATCTATAGCTATATCAAATTCACAAAACAAAGATCAGAACCATTCGGCCAAAAGTGGTGGAAGTGGCTGTTCTTTACAGGGGTGTCTTTATCTTGCGTCATAAGTACCAAGTATGTGGGGGTGTTCACCTATCTTACAATAGGCTGTGGTGTCCTGTTTGACTTATGGAGTTTACTGGATTATAAAAAGGGACATTCCTTGGCATATGTTGGTAAACACTTTGCTGCACGATTTTTCCTTCTAATACTGGTCCCTTTCTTGATATATCTCAATTGGTTTTATGTTCATTTCGCTATTCTAAGCAAGTCTGGCCCAGGAGACAGTTTTATGAGCTCTGAATTCCAGGAGACTCTCGGAGATTCTCCTCTTGCAGCTTTCGCAAAGGAAGTTCACTTTAACGACATAATCACAATAAAGCATAAAGAGACTGATGCCATGTTGCACTCACACTTGGCAAACTACCCCCTCCGTTACGAGGACGGGAGGGTATCATCTCAAGGTCAACAAGTTACAGCATACTCTGGAGAGGACCCAAACAATAATTGGCAGATTATTTCTCCCGAAGGACTTACTGGCGTTGTAACTCAGGGCGATGTCGTTAGACTGAGACACGTTGGGACAGATGGCTATCTACTGACGCATGATGTTGCGTCTCCTTTCTATCCAACTAACGAGGAGTTTACTGTAGTGGGACAGGAGAAAGCTACTCAACGCTGGAACGAAACACTTTTTAGAATTGATCCCTATGACAAGAAGAAAACCCGTCCTTTGAAGTCGAAAGCTTCATTTTTCAAACTCATTCATGTTCCTACGGTTGTGGCCATGTGGACTCATAATGACCAGCTTCTTCCTGATTGGGGTTTCAACCAACAAGAAGTCAATGGTAATAAGAAGCTTGCTGATGAATCAAACTTATGGGTTGTAGACAATATCGTCGATATTGCAGAGGACGATCCAAGGAAACACTACGTTCCAAAGGAAGTGAAAAATTTGCCATTTTTGACCAAGTGGTTGGAATTACAAAGACTTATGTTTATTCAGAATAACAAGTTGAGCTCAGATCATCCATTTGCGTCTGACCCTATATCTTGGCCTTTTTCACTTAGTGGGGTTTCATTTTGGACAAACAACGAGTCACGCAAACAGATCTATTTTGTCGGAAATATTCCTGGATGGTGGATGGAGGTTGCAGCATTGGGATCCTTTCTAGGACTCGTGTTTGCAGATCAGTTCACGAGAAGAAGAAACAGTCTTGTTTTGACCAATAGCGCCAGGTCTCGGTTATACAATAATTTGGGGTTCTTCTTTGTAGGCTGGTGTTGTCATTACCTACCCTTTTTCCTAATGAGCCGTCAAAAATTTTTGCACCATTACTTACCTGCACATTTAATAGCAGCCATGTTCACTGCTGGTTTCTTGGAATTTATTTTTACTGACAACAGAACTGAAGAATTCAAGGATCAGAAAACTTCATGTGAACCTAACTCTAATTCTTCAAAGCCGAAAGAGCAATTGATTCTGTGGTTAAGTTTCTCGTCCTTTGTCGCTTTGCTACTAAGCATCATTGTTTGGACTTTCTTCTTTTTTGCTCCTCTAACATATGGTAATACTGCGCTTTCGGCGGAGGAGGTTCAGC

AGTATACAATGTGTAGTTCAACGCAAAGGAAATTCTAACTTTCTGTGCAATCTGGTGACAATTTCTAAATAACTATCACAATTGGAAGAAGAGATTATCCCAAATCTTATCAAAAAATCGATGATTGCCAGTGCACAATTAGGCTTGAATTTTTCTTGCAGCAACGAAGAGATTACTTCAGTGATGTTCATTAGCCTGAAATCTTCACTTTCGTGGTCTATCGGATTAGGAATTAGACCTTGTTTCATCGGCAGGTCGTATATGTATTCCACTTCTGGTTGAATAAAATCTTCGGGTGGTTTGTTTCTGAACATATATGAGATGGCTCCCACTGGACTGATATATTGCGAAACATAGTCCTCATTCAAACCCTGCCTCCTCGTAACATTCTTTCAGGCAAGTTTGCAAAGTGCCATTAGGATATTCCAAGCCTCCTGCCACAGTATTATCTAACATACCGGGAAATGTTGGTTTGTGTCTGCTTCTCCTAGGTATCCAAAGTTGAATACTGTTAGGATCGGCAGAATTTTGCAAATATCCATTGATATGAACTCCATAAGTAACAACTCCCAAAATATTAGAAAAGCCCTTTCCACCAACATGTACATCTTATGGTTATCGCAGTAAACTGCAAAAAGCTCATTTCTCCAACCGCTAAGGGTTTCAAAGAGACGCTGATCTCTCCAACGCTGAGCTATCTTTGCAAACATCTGCGTTCTTTTATTTTCGCTATCCAGACTAGGAATTATCTTGACTTCGTGTTTTTCATTATTTACTATCACAGCCTGTGTTTCGAACTCAAATTGTTTTGCCACCTTGGGAATTATATACCCTAGTAAGATCCCATCATGCGATAAGAATTTATACACAGATACTTCAAATTCATGAAAAGATGGCTCATCTTTATGAGGAACAGAATCAACAGATCTGACTAGATCAATATATGGCATTGGTTGATTTTATTCAATGGTTATCTATCTCAAACATGCTATAAAAATAAGGTAATTCCTTTATGGTGTTAGGGTGTTATAGTTTTTGCGTAGAAAATAATTGTCATCATTTTTGGGCAACCTATGAAACAACTACTCAGAGAAGTTGAGACATCTCTTTTGACAAATGAAACCGAAATATCCCCTGCCCTTAAGCTATTAATTACTCAGTTAAATAAATCAACCCATGAAGATAAATCAACAGAAAGAAAAACGTTTTGGCTAGCATTAGACAATTTAAGGCAAAAAATCGGTCTACAATCCCAATCACATGTCCTTTTCTTTCTACATCTTTTTGAAGAGCTAGCTCCAACTTTAGAAAATGAGAAAATATTTTTAACCTGGATTACTTCTTTTTTGAAGTTAGCAATTAATAGTGCAGGGGTACCACATTGTGTGGTGAACGAGTCAAGGAGAATTATAATGAATTTATTATTGCCCTCAAAAGCTACAAACACCGAATACAATTTGTTAAAGAATTCTGCTGCAGGCATTCAATTACTTGTGCAAGTGTATTTGCTAAAAACTGATTTAGTTGTTGATTCCACTTCTAGTAGTCCCCAGGAGTATGAAGAGAGGGTTAGATTCATAAAGAAAAACTGCAGGGATTTACTACAAGGTCTTGATTTAAATAATCAAGTACTAGAGGCTATCAGCAAAGAATTTACGGATCCTCACTACCGCTTCGAGTGCTTCGTACTTTTGTCCTCATTAATGTCGTCATCAGCCTTGTTGTACCAGATAATGCAAACAACTTTGTGGCATAATATACTTTTGTCTATATTGATAGATAAAAGTAACAGTGTGGTTGAGTCAGGAATCAAGGTTCTCAGTATGGTTTTGCCCCACGTCTGTGATGTAATAGCGGATTATCTACCGACCATTATGGCGATTTTAAGTAAAGGTCTGGGGGGTGTTGAAATTGATGATGAGTCACCATTACCATCAAATTGGAAAGTATTGAATGATCAGGATCCTGAAATTATTGGTCCAGCATTTGTTAGCTATAAACAACTGTTCACTGTATTATACGGCCTGTTCCCTCTTAGTTTAACATCATTATTCGCAGTCCATCTACATATATCGACTCTAACAAGATTATAGACGATCTCAAGCTTCAGTTGCTTGAAACTAAAGTGAAGTCAAAGTGTCAGGACTTGCTAAAGTGTTTTATTGTTCATCCAAATTATTTTATATATTCTTCCCAGGAGGAAGAAATTTTTGATACTTCAAGGTGGGACAAAATGCACTCCCCGAACGAGTAGCAGCATTTTGTTATCAATTGGAATTCCGTGGGACATCGAAGGAGAATGCCTTTGATATGAGGGTAGATGACCTTTTGGAAGGTCATCGATATCTATATTTGAAAGATATGAAGGATGCGCAGAAAGAGAGGGCTAAAAAATGTGAAAATTCTATTATCTCACTCGAAAGTTCATCTGATAGTAAGTCAGTTTCACAATACGACGAAGACTCGACGAAAGAAACCACTTGCAGGCATGTTTCGTTTTATTTAAGAGAGATCCTTTTGGCAAAAAATGAATTGGACTTCACGCTACATATCAATCAGGTACTTGGAGCCGAGTGTGAGCTTTTGAAAAAAAAATTGAACGAAATGGATACCCTACGAGATCAAAACAGGTTTTTAGCTGACATAAACGAAGGTTACGAATACAGCAATCTAAGGCGAGTGAGCAAATTACGGAATTGCTCAAAGAAAAAGAGCGTTCTCAAAATGATTTCAACTCTCTGGTTACTCATATGCTTAAACAATCTAACGAATTAAAAGAAAGGGAGTCGAAACTAGTCGAGATTCATCAATCAATGATGCAGAGATAGGAGATTTAAATTATAGGTTGGAAAAACTGTGCAACCTTATACAACCCAAAGAATTAGAAGTGGAACTGCTCAAGAAGAAGTTGCGTGTAGCATCGATCCTTTTTTCGCAAGATAAATCAAAATCTTCAAGCAAGACATCTCTAGCACATTT GCACCAGGCAGGCGACGCAACT 50PpPMT4 MIKSRKRSRKVSLNTEKELKNSHISLGDERWYTVGLLLVTITAFC (protein)TRFYAINYPDEVVFDEVHFGKFASYYLERTYFFDLHPPFAKLLIAFVGFLAGYNGEFKFTTIGESYIKNEVPYVVYRSLSAVQGSLTVPIVYLCLKECGYTVLTCVFGACIILFDGAHVAETRLILLDATLIFFVSLSIYSYIKFTKQRSEPFGQKWWKWLEFTGVSLSCVISTKYVGVFTYLTIGCGVLFDLWSLLDYKKGHSLAYVGKHFAARFFLLILVPFLIYLNWFYVHFAILSKSGPGDSFMSSEFQETLGDSPLAAFAKEVHFNDIITIKHKETDAMLHSHLANYPLRYEDGRVSSQGQQVTAYSGEDPNNNWQIISPEGLTGVVTQGDVVRLRHVGTDGYLLTHDVASPFYPTNEEFTVVGQEKATQRWNETLFRIDPYDKKKTRPLKSKASFFKLIHVPTVVAMWTHNDQLLPDWGFNQQEVNGNKKLADESNLWVVDNIVDIAEDDPRKHYVPKEVKNLPFLTKWLELQRLMFIQNNKLSSDHPFASDPISWPFSLSGVSFWINNESRKQIYFVGNIPGWWMEVAALGSFLGLVFADQFTRRRNSLVLTNSARSRLYNNLGFFFVGWCCHYLPFFLMSRQKFLHHYLPAHLIAAMFTAGFLEFIFTDNRTEEFKDQKTSCEPNSNSSKPKEQLILWLSESSFVALLLSIIVWTFFFFAPLTYGNTALSAEEVQQRQWLDMKLQFAK 51 anti-DKK1ACGATGGTCGCTTGGTGGTCTTTGTTTCTGTACGGTCTTCAGG Heavy chainTCGCTGCACCTGCTTTGGCTGAGGTTCAGTTGGTTCAATCTGG (VH +TGCTGAGGTTAAGAAACCTGGTGCTTCCGTTAAGGTTTCCTGT IgG2m4) (α-AAGGCTTCCGGTTACACTTTCACTGACTACTACATCCACTGGG amylaseTTAGACAAGCTCCAGGTCAAGGATTGGAATGGATGGGATGGA encodingTTCACTCTAACTCCGGTGCTACTACTTACGCTCAGAAGTTCCA sequencesGGCTAGAGTTACTATGTCCAGAGACACTTCTTCTTCCACTGCT underlined)TACATGGAATTGTCCAGATTGGAATCCGATGACACTGCTATGT (DNA)ACTTTTGTTCCAGAGAGGACTACTGGGGACAGGGAACTTTGGTTACTGTTTCCTCCGCTTCTACTAAAGGGCCCTCTGTTTTTCCATTGGCTCCATGTTCTAGATCCACTTCCGAATCCACTGCTGCTTTGGGATGTTTGGTTAAGGACTACTTCCCAGAGCCAGTTACTGTTTCTTGGAACTCCGGTGCTTTGACTTCTGGTGTTCACACTTTCCCAGCTGTTTTGCAATCTTCCGGTTTGTACTCCTTGTCCTCCGTTGTTACTGTTACTTCCTCCAACTTCGGTACTCAGACTTACACTTGTAACGTTGACCACAAGCCATCCAACACTAAGGTTGACAAGACTGTTGAGAGAAAGTGTTGTGTTGAGTGTCCACCATGTCCAGCTCCACCAGTTGCTGGTCCATCCGTTTTTTTGTTCCCACCAAAGCCAAAGGACACTTTGATGATCTCCAGAACTCCAGAGGTTACATGTGTTGTTGTTGACGTTTCCCAAGAGGACCCAGAGGTTCAATTCAACTGGTACGTTGACGGTGTTGAAGTTCACAACGCTAAGACTAAGCCAAGAGAAGAGCAGTTCAACTCCACTTTCAGAGTTGTTTCCGTTTTGACTGTTTTGCACCAGGATTGGTTGAACGGTAAAGAATACAAGTGTAAGGTTTCCAACAAGGGATTGCCATCCTCCATCGAAAAGACTATCTCCAAGACTAAGGGACAACCAAGAGAGCCACAGGTTTACACTTTGCCACCATCCAGAGAAGAGATGACTAAGAACCAGGTTTCCTTGACTTGTTTGGTTAAAGGATTCTACCCATCCGACATTGCTGTTGAGTGGGAATCTAACGGTCAACCAGAGAACAACTACAAGACTACTCCACCAATGTTGGATTCTGACGGTTCCTTCTTCTTGTACTCCAAGTTGACTGTTGACAAGTCCAGATGGCAACAGGGTAACGTTTTCTCCTGTTCCGTTATGCATGAGGCTTTGCACAACCACTACACTCAAAAGTCCTTGTCTTTGTCC CCTGGTAAGTAA 52 anti-DKK1EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQAPGQ Heavy chainGLEWMGWIHSNSGATTYAQKFQARVTMSRDTSSSTAYMELSRL (VH +ESDDTAMYFCSREDYWGQGTLVTVSSASTKGPSVFPLAPCSRST IgG2m4)SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL (protein)YSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 53 anti-DKK1ACGATGGTCGCTTGGTGGTCTTTGTTTCTGTACGGTCTTCAGG Light chainTCGCTGCACCTGCTTTGGCTCAGTCCGTTTTGACACAACCACC (VL + lambdaATCTGTTTCTGGTGCTCCAGGACAGAGAGTTACTATCTCCTGT constantACTGGTTCCTCTTCCAACATTGGTGCTGGTTACGATGTTCACT regions) (α-GGTATCAACAGTTGCCAGGTACTGCTCCAAAGTTGTTGATCTA amylaseCGGTTACTCCAACAGACCATCTGGTGTTCCAGACAGATTCTCT encodingGGTTCTAAGTCTGGTGCTTCTGCTTCCTTGGCTATCACTGGAG sequencesTGAGACCAGATGACGAGGCTGACTACTACTGTCAATCCTACG underlined)ACAACTCCTTGTCCTCTTACGTTTTCGGTGGTGGTACTCAGTT (DNA)GACTGTTTTGTCCCAGCCAAAGGCTAATCCAACTGTTACTTTGTTCCCACCATCTTCCGAAGAACTGCAGGCTAATAAGGCTACTTTGGTTTGTTTGATCTCCGACTTCTACCCAGGTGCTGTTACTGTTGCTTGGAAGGCTGATGGTTCTCCAGTTAAGGCTGGTGTTGAGACTACTAAGCCATCCAAGCAGTCCAATAACAAGTACGCTGCTAGCTCTTACTTGTCCTTGACACCAGAACAATGGAAGTCCCACAGATCCTACTCTTGTCAGGTTACACACGAGGGTTCTACTGTTGAAAAGACTGTTGCTCCAACTGAGTGTTCCTAA 54 anti-DKK1QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGT Light chainAPKLLIYGYSNRPSGVPDRFSGSKSGASASLAITGLRPDDEADYY (VL + lambdaCQSYDNSLSSYVFGGGTQLTVLSQPKANPTVTLFPPSSEELQANK constantATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKY regions)AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (protein) 55 Human BiPGAGGAAGAGGACAAGAAAGAGGATGTTGGTACTGTTGTCGGT (DNA)ATCGACTTGGGTACTACCTACTCCTGTGTCGGTGTTTTCAAGAACGGTAGAGTGGAGATTATCGCCAACGACCAGGGTAACAGAATTACTCCATCCTACGTTGCTTTTACCCCAGAAGGAGAGAGATTGATCGGAGACGCTGCTAAGAACCAATTGACCTCCAACCCAGAGAACACTGTTTTCGACGCCAAGAGACTGATTGGTAGAACTTGGAACGACCCATCCGTTCAACAAGACATCAAGTTCTTGCCCTTCAAGGTCGTCGAGAAGAAAACCAAGCCATACATCCAGGTTGACATCGGTGGTGGTCAAACTAAGACTTTCGCTCCAGAGGAAATCTCCGCTATGGTCCTGACTAAGATGAAAGAGACTGCCGAGGCTTACTTGGGTAAAAAGGTTACCCACGCTGTTGTTACTGTTCCAGCTTACTTCAACGACGCTCAGAGACAAGCTACTAAGGACGCTGGTACTATCGCTGGACTGAACGTGATGAGAATCATCAACGAGCCAACTGCTGCTGCTATTGCCTACGGATTGGACAAGAGAGAGGGAGAGAAGAACATCTTGGTTTTCGACTTGGGTGGTGGTACTTTCGACGTTTCCTTGTTGACCATCGACAACGGTGTTTTCGAAGTTGTTGCTACCAACGGTGATACTCACTTGGGTGGAGAGGACTTCGATCAGAGAGTGATGGAACACTTCATCAAGCTGTACAAGAAGAAAACCGGAAAGGACGTTAGAAAGGACAACAGAGCCGTTCAGAAGTTGAGAAGAGAGGTTGAGAAGGCTAAGGCTTTGTCCTCCCAACACCAAGCTAGAATCGAGATCGAATCCTTCTACGAGGGTGAAGATTTCTCCGAGACCTTGACTAGAGCCAAGTTCGAAGAGCTGAACATGGACCTGTTCAGATCCACTATGAAGCCAGTTCAGAAGGTTTTGGAGGATTCCGACTTGAAGAAGTCCGACATCGACGAGATTGTTTTGGTTGGTGGTTCCACCAGAATCCCAAAGATCCAGCAGCTGGTCAAAGAGTTCTTCAACGGTAAAGAGCCATCCAGAGGTATTAACCCAGATGAGGCTGTTGCTTACGGTGCTGCTGTTCAAGCTGGTGTTTTGTCTGGTGACCAGGACACTGGTGACTTGGTTTTGTTGCATGTTTGCCCATTGACTTTGGGTATCGAGACTGTTGGTGGTGTTATGACCAAGTTGATCCCATCCAACACTGTTGTTCCCACCAAGAACTCCCAAATTTTCTCCACTGCTTCCGACAACCAGCCAACCGTTACTATTAAGGTCTACGAAGGTGAAAGACCATTGACCAAGGACAACCACTTGTTGGGAACTTTCGACTTGACTGGTATTCCACCTGCTCCAAGAGGTGTTCCACAAATCGAGGTTACCTTCGAGATCGACGTCAACGGTATCTTGAGAGTTACTGCCGAGGATAAGGGAACCGGTAACAAGAACAAGATCACCATCACCAACGACCAAAACAGATTGACCCCCGAAGAGATCGAAAGAATGGTCAACGATGCTGAGAAGTTCGCCGAAGAGGATAAGAAGCTGAAAGAGAGAATCGACACCAGAAACGAGTTGGAATCCTACGCTTACTCCTTGAAGAACCAGATCGGTGACAAAGAAAAGTTGGGTGGAAAGCTGTCATCCGAAGATAAAGAAACTATGGAAAAGGCCGTCGAAGAAAAGATTGAGTGGCTGGAATCTCACCAAGATGCTGACATCGAGGACTTCAAGGCCAAGAAGAAAGAGTTGGAAGAGATCGTCCAGCCAATCATTTCTAAGTTGTACGGTTCTGCTGGTCCACCACCAACTGGTGAAGAAGATACTGCCGAGCACGACGA GTTGTAG 56 Human BiPEEEDKKEDVGTVVGIDLGTTYSCVGVFKNGRVEIIANDQGNRITP (protein)SYVAFTPEGERLIGDAAKNQLTSNPENTVFDAKRLIGRTWNDPS ATPaseVQQDIKFLPFKVVEKKTKPYIQVDIGGGQTKTFAPEEISAMVLTK domainMKETAEAYLGKKVTHAVVTVPAYFNDAQRQATKDAGTIAGLN underlinedVMRIINEPTAAAIAYGLDKREGEKNILVFDLGGGTFDVSLLTIDNGVFEVVATNGDTHLGGEDFDQRVMEHFIKLYKKKTGKDVRKDNRAVGKLRREVEKAKALSSQHQARIEIESFYEGEDFSETLTRAKFEELNMDLFRSTMKPVQKVLEDSDLKKSDIDEIVLVGGSTRIPKIQQLVKEFFNGKEPSRGINPDEAVAYGAAVQAGVLSGDQDTGDLVLLHVCPLTLGIETVGGVMTKLIPSNTVVPTKNSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLGTFDLTGIPPAPRGVPQIEVTFEIDVNGILRVTAEDKGTGNKNKITITNDQNRLTPEEIERMVNDAEKFAEEDKKLKERIDTRNELESYAYSLKNQIGDKEKLGGKLSSEDKETMEKAVEEKIEWLESHQDADIEDFKAKKKELEEIVQPIISKLYGSAGPP PTGEEDTAEHDEL 57Chimeric BiP GACGATGTCGAATCTTATGGAACAGTGATTGGTATCGATTTG (DNA)GGTACCACGTACTCTTGTGTCGGTGTGATGAAGTCGGGTCGTGTAGAAATTCTTGCTAATGACCAAGGTAACAGAATCACTCCTTCCTACGTTAGTTTCACTGAAGACGAGAGACTGGTTGGTGATGCTGCTAAGAACTTAGCTGCTTCTAACCCAAAAAACACCAATCTTTGATATTAAGAGATTGATCGGTATGAAGTATGATGCCCCAGAGGTCCAAAGAGACTTGAAGCGTCTTCCTTACACTGTCAAGAGCAAGAACGGCCAACCTGTCGTTTCTGTCGAGTACAAGGGTGAGGAGAAGTCTTTCACTCCTGAGGAGATTTCCGCCATGGTCTTGGGTAAGATGAAGTTGATCGCTGAGGACTACTTAGGAAAGAAAGTCACTCATGCTGTCGTTACCGTTCCAGCCTACTTCAACGACGCTCAACGTCAAGCCACTAAGGATGCCGGTCTCATCGCCGGTTTGACTGTTCTGAGAATTGTGAACGAGCCTACCGCCGCTGCCCTTGCTTACGGTTTGGACAAGACTGGTGAGGAAAGACAGATCATCGTCTACGACTTGGGTGGAGGAACCTTCGATGTTTCTCTGCTTTCTATTGAGGGTGGTGCTTTCGAGGTTCTTGCTACCGCCGGTGACACCCACTTGGGTGGTGAGGACTTTGACTACAAGAGTTGTTCGCCACTTCGTTAAGATTTTCAAGAAGAAGCATAACATTGACATCAGCAACAATGATAAGGCTTTAGGTAAGCTGAAGAGAGAGGTCGAAAAGGCCAAGCGTACTTTGTCTTCCCAGATGACTACCAGAATTGAGATTGACTCTTTCGTCGACGGTATCGACTTCTCTGAGCAACTGTCTAGAGCTAAGTTTGAGGAGATCAACATTGAATTATTCAGAAGACACTGAAACCAGTTGAACAAGTCCTCAAAGACGCTGGTGTCAAGAAATCTGAAATTGATGACATTGTCTTGGTTGGTGGTTCTACCAGATTCCAAAGGTTCAACAATTATTGGAGGATTACTTTGACGGAAAGAAGGCTTCTAAGGGAATTAACCCAGATGAAGCTGTCGCATACGGTGCTGCTGTTCAGGCTGGTGTTTTGTCTGGTGATCAAGATACAGGTGACCTGGTACTGCTTGATGTATGTCCCCTTACACTTGGTATTGAAACTGTGGGAGGTGTCATGACCAAACTGATTCCAAGGAACACAGTGGTGCCTACCAAGAAGTCTCAGATCTTTTCTACAGCTTCTGATAATCAACCAACTGTTACAATCAAGGTCTATGAAGGTGAAAGACCCCTGACAAAAGACAATCATCTTCTGGGTACATTTGATCTGACTGGAATTCCTCCTGCTCCTCGTGGGGTCCCACAGATTGAAGTCACCTTTGAGATAGATGTGAATGGTATTCTTCGAGTGACAGCTGAAGACAAGGGTACAGGGAACAAAAATAAGATCACAATCACCAATGACCAGAATCGCCTGACACCTGAAGAAATCGAAAGGATGGTTAATGATGCTGAGAAGTTTGCTGAGGAAGACAAAAAGCTCAAGGAGCGCATTGATACTAGAAATGAGTTGGAAAGCTATGCCTATTCTCTAAAGAATCAGATTGGAGATAAAGAAAAGCTGGGAGGTAAACTTTCCTCTGAAGATAAGGAGACCATGGAAAAAGCTGTAGAAGAAAAGATTGAATGGCTGGAAAGCCACCAAGATGCTGACATTGAAGACTTCAAAGCTAAGAAGAAGGAACTGGAAGAAATTGTTCAACCAATTATCAGAAACTCTATGGAAGTGCAGGCCCTCCCCCAACTGGTGAAGAGGATACAGCAGAACATGATGAGTTGTAG 58 Chimeric BiPDDVESYGTVIGIDLGTTYSCVGVMKSGRVEILANDQGNRITPSYV (protein)SFTEDERLVGDAAKNLAASNPKNTIFDIKRLIGMKYDAPEVQRD ATPaseLKRLPYTVKSKNGQPVVSVEYKGEEKSFTPEEISAMVLGKMKLI domainAEDYLGKKVTHAVVTVPAYENDAQRQATKDAGLIAGLTVLRIV underlinedNEPTAAALAYGLDKTGEERQIIVYDLGGGTFDVSLLSIEGGAFEVLATAGDTHLGGEDFDYRVVRHFVKIFKKKHNIDISNNDKALGKLKREVEKAKRTLSSQMTTRIEIDSFVDGIDFSEQLSRAKFEEINIELFKKTLKPVEQVLKDAGVKKSEIDDIVLVGGSTRIPKVQQLLEDYFDGKKASKGINPDEAVAYGAAVQAGVLSGDQDTGDLVLLDVCPLTLGIETVGGVMTKLIPRNTVVPTKKSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLGTFDLTGIPPAPRGVPQIEVTFEIDVNGILRVTAEDKGTGNKNKITITNDQNRLTPEEIERMVNDAEKFAEEDKKLKERIDTRNELESYAYSLKNQIGDKEKLGGKLSSEDKETMEKAVEEKIEWLESHQDADIEDFKAKKKELEEIVQPIISKLYGSAGPPPTGEEDT AEHDEL 59 PpPDI1AACACGAACACTGTAAATAGAATAAAAGAAAACTTGGATAGT promoterAGAACTTCAATGTAGTGTTTCTATTGTCTTACGCGGCTCTTTAGATTGCAATCCCCAGAATGGAATCGTCCATCTTTCTCAACCCACTCAAAGATAATCTACCAGACATACCTACGCCCTCCATCCCAGCACCACGTCGCGATCACCCCTAAAACTTCAATAATTGAACACGTACTGATTTCCAAACCTTCTTCTTCT TCCTATCTATAAGA 60 PpPMR1ATGACAGCTAATGAAAATCCTTTTGAGAATGAGCTGACAGGATCTGAATCTGCCCCCCCTGCATTGGAATCGAAGACTGGAGAGTCTCTTAAGTATTGCAAATATACCGTGGATCAGGTCATAGAAGAGTTTCAAACGGATGGTCTCAAAGGATTGTGCAATTCCCAGGACATCGTATATCGGAGGTCTGTTCATGGGCCAAATGAAATGGAAGTCGAAGAGGAAGAGAGTCTTTTTTCGAAATTCTTGTCAAGTTTCTACAGCGATCCATTGATTCTGTTACTGATGGGTTCCGCTGTGATTAGCTTTTTGATGTCTAACATTGATGATGCGATATCTATCACTATGGCAATTACGATCGTTGTCACAGTTGGATTTGTTCAAGAGTATCGATCCGAGAAATCATTGGAGGCATTGAACAAGTTAGTCCCTGCCGAAGCTCATCTAACTAGGAATGGGAACACTGAAACTGTTCTTGCTGCCAACCTAGTCCCAGGAGACTTGGTGGATTTTTCGGTTGGTGACAGAATTCCGGCTGATGTGAGAATTATTCACGCTTCCCACTTGAGTATCGACGAGAGCAACCTAACTGGTGAAAATGAACCAGTTTCTAAAGACAGCAAACCTGTTGAAAGTGATGACCCAAACATTCCCTTGAACAGCCGTTCATGTATTGGGTATATGGGCACTTTAGTTCGTGATGGTAATGGCAAAGGTATTGTCATCGGAACAGCCAAAAACACAGCTTTTGGCTCTGTTTTCGAAATGATGAGCTCTATTGAGAAACCAAAGACTCCTCTTCAACAGGCTATGGATAAACTTGGTAAGGATTTGTCTGCTTTTTCCTTCGGAATCAATCGGCCTTATTTGCTTGGTTGGTGTTTTTCAAGGTAGACCCTGGTTGGAAATGTTCCAGATCTCTGTATCCTTGGCTGTTGCTGCGATTCCAGAAGGTCTTCCTATTATTGTGACTGTGACTCTTGCTCTTGGTGTGTTGCGTATGGCTAAACAGAGGGCCATCGTCAAAAGACTGCCTAGTGTTGAAACTTTGGGATCCGTCAATGTTATCTGTAGTGATAAGACGGGAACATTGACCCAAAATCATATGACCGTTAACAGATTATGGACTGTGGATATGGGCGATGAATTCTTGAAAATTGAACAAGGGGAGTCCTATGCCAATTATCTCAAACCCGATACGCTAAAAGTTCTGCAAACTGGTAATATAGTCAACAATGCCAAATATTCAAATGAAAAGGAAAAATACCTCGGAAACCCAACTGATATTGCAATTATTGAATCTTTAGAAAAATTTGATTTGCAGGACATTAGAGCAACAAAGGAAAGAATGTTGGAGATTCCATTTTCTTCGTCCAAGAAATATCAGGCCGTCAGTGTTCACTCTGGAGACAAAAGCAAATCTGAAATTTTTGTTAAAGGCGCTCTGAACAAAGTTTTGGAAAGATGTTCAAGATATTACAATGCTGAAGGTATCGCCACTCCACTCACAGATGAAATTAGAAGAAAATCCTTGCAATGGCCGATACGTTAGCATCTTCAGGATTGAGAATACTGTCGTTTGCTTACGACAAAGGCAATTTTGAAGAAACTGGCGATGGACCATCGGATATGAATCTTTTGTGGTCTTTTAGGTATGAACGATCCTCCTAGACCATCTGTAAGTAAAATCAATTTTGAAATTCATGAGAGGTGGGGTTCACATTATTATGATTACAGGAGATTCAGAATCCACGGCCGTAGCCGTTGCCAAACAGGTCGGAATGGTAATTGACAATTCAAAATATGCTGTCCTCAGTGGAGACGATATAGATGCTATGAGTACAGAGCAACTGTCTCAGGCGATCTCACATTGTTCTGTATTTGCCCGGACTACTCCAAAACATAAGGTGTCCATTGTAAGAGCACTACAGGCCAGAGGAGATATTGTTGCAATGACTGGTGACGGTGTCAATGATGCCCCAGCTCTAAAACTGGCCGACATCGGAATTGCCATGGGTAATATGGGGACCGATGTTGCCAAAGAGGCAGCCGACATGGTTTTGACTGATGATGACTTTTCTACAATCTTATCTGCAATCCAGGAGGGTAAAGGTATTTTCTACAACATCCAGAACTTTTTAACGTTCCAACTTTCTACTTCAATTGCTGCTCTTTCGTTAATTGCTCTGAGTACTGCTTTCAACCTGCCAAAATCCATTGAATGCCATGCAGATTTTGTGGATCAATATTATCATGGATGGACCTCCAGCTCAGTCTTTGGGTGTTGAGCCAGTTGATAAAGCTGTGATGAACAAACCACCAAGAAAGCGAAATGATAAAATTCTGACAGGTAAGGTGATTCAAAGGGTAGTACAAAGTAGTTTTATCATTGTTTGTGGTACTCTGTACGTATACATGCATGAGATCAAAGATAATGAGGTCACAGCAAGAGACACTACGATGACCTTTACATGCTTTGTATTCTTTGACATGTTCAACGCATTAACGACAAGACACCATTCTAAAAGTATTGCAGAACTTGGATGGAATAATACTAGTTCAACTTTTCCGTTGCAGCTTCTATTTTGGGTCAACTAGGAGCTATTTACATTCCATTTTTGCAGTCTATTTTCCAGACTGAACCTCTGAGCCTCAAAGATTTGGTCCATTTATTGTTGTTATCGAGTTCAGTATGGATTGTAGACGAGCTTCGAAAACTCTACGTCAGGAGACGTGACGCATCCCCATACAATGGATACAGCA TGGCTGTTTGA 61 PpPMR1MTANENPFENELTGSSESAPPALESKTGESLKYCKYTVDQVIEEFQTDGLKGLCNSQDIVYRRSVHGPNEMEVEEEESLFSKFLSSFYSDPLILLLMGSAVISFLMSNIDDAISITMAITIVVTVGFVQEYRSEKSLEALNKLVPAEAHLTRNGNTETVLAANLVPGDLVDFSVGDRIPADVRIIHASHLSIDESNLTGENEPVSKDSKPVESDDPNIPLNSRSCIGYMGTLVRDGNGKGIVIGTAKNTAFGSVFEMMSSIEKPKTPLQQAMDKLGKDLSAFSFGIIGLICLVGVFQGRPWLEMFQISVSLAVAAIPEGLPIIVTVTLALGVLRMAKQRAIVKRLPSVETLGSVNVICSDKTGTLTQNHMTVNRLWTVDMGDEFLKIEQGESYANYLKPDTLKVLQTGNIVNNAKYSNEKEKYLGNPTDIAIIESLEKFDLQDIRATKERMLEIPFSSSKKYQAVSVHSGDKSKSEIFVKGALNKVLERCSRYYNAEGIATPLTDEIRRKSLQMADTLASSGLRILSFAYDKGNFEETGDGPSDMIFCGLLGMNDPPRPSVSKSILKFMRGGVHIIMITGDSESTAVAVAKQVGMVIDNSKYAVLSGDDIDAMSTEQLSQAISHCSVFARTTPKHKVSIVRALQARGDIVAMTGDGVNDAPALKLADIGIAMGNMGTDVAKEAADMVLTDDDFSTILSAIQEGKGIFYNIQNFLTFQLSTSIAALSLIALSTAFNLPNPLNAMQILWINIIMDGPPAQSLGVEPVDKAVMNKPPRKRNDKILTGKVIQRVVQSSFIIVCGTLYVYMHEIKDNEVTARDTTMTFTCFVFFDMFNALTTRHHSKSIAELGWNNTMFNFSVAASILGQLGAIYIPFLQSIFQTEPLSLKDLVHLLLLSSSVWIVDE LRKLYVRRRDASPYNGYSMAV 62Arabidopsis ATGGGAAAGGGTTCCGAGGACCTGGTTAAGAAAGAATCCCTG ThalianaAACTCCACTCCAGTTAACTCTGACACTTTCCCAGCTTGGGCTA AtECA1AGGATGTTGCTGAGTGCGAAGAGCACTTCGTTGTTTCCAGAG (codonAGAAGGGTTTGTCCTCCGACGAAGTCTTGAAGAGACACCAAA optimized TCTACGGACTGAACGAGTTGGAAAAGCCAGAGGGAACCTCCA forTCTTCAAGCTGATCTTGGAGCAGTTCAACGACACCCTTGTCAG PichiaAATTTTGTTGGCTGCCGCTGTTATTTCTTCGTCCTGGCTTTTT pastoris)TTGATGGTGACGAGGGTGGTGAAATGGGTATCACTGCCTTCGTTGAGCCTTTGGTCATCTTCCTGATCTTGATCGTTAACGCCATCGTTGGTATCTGGCAAGAGACTAACGCTGAAAAGGCTTTGGAGGCCTTGAAAGAGATTCAATCCCAGCAGGCTACCGTTATGAGAGATGGTACTAAGGTTTCCTCCTTGCCAGCTAAAGAATTGGTTCCAGGTGACATCGTTGAGCTGAGAGTTGGTGATAAGGTTCCAGCCGACATGAGAGTTGTTGCTTTGATCTCCTCCACCTTGAGAGTTGAACAAGGTTCCCTGACTGGTGAATCTGAGGCTGTTTCCAAGACTACTAAGCACGTTGACGAGAACGCTGACATCCAGGGTAAAAAGTGCATGGTTTTCGCCGGTACTACCGTTGTTAACGGTAACTGCATCGTTTGGTCACTGACACTGGAATGAACACCGAGATCGGTAGAGTTCACTCCCAAATCCAAGAAGCTGCTCAACACGAAGAGGACACCCCATTGAAGAAGAAGCTGAACGAGTTCGGAGAGGTCTTGACCATGATCATCGGATTGATCTGTGCCCTGGTCTGGTTGATCAACGTCAAGTACTTCTTGTCCTGGGAATACGTTGATGGATGGCCAAGAAACTTCAAGTTCTCCTTCGAGAAGTGCACCTACTACTTCGAGATCGCTGTTGCTTTGGCTGTTGCTGCTATTCCAGAGGGATTGCCAGCTGTTATCACCACTTGCTTGGCCTTGGGTACTAGAAAGATGGCTCAGAAGAACGCCCTTGTTAGAAAGTTGCCATCCGTTGAGACTTTGGGTTGTACTACCGTCATCTGTTCCGACAAGACTGGTACTTTGACTACCAACCAGATGGCCGTTTCCAAATTGGTTGCCATGGGTTCCAGAATCGGTACTCTGAGATCCTTCAACGTCGAGGGAACTTCTTTTGACCCAAGAGATGGAAAGATTGAGGACTGGCCAATGGGTAGAATGGACGCCAACTTGCAGATGATTGCTAAGATCGCCGCTATCTGTAACGACGCTAACGTTGAGCAATCCGACCAACAGTTCGTTTCCAGAGGAATGCCAACTGAGGCTGCCTTGAAGGTTTTGGTCGAGAAGATGGGTTTCCCAGAAGGATTGAACGAGGCTTCTTCCGATGGTGACGTCTTGAGATGTTGCAGACTGTGGAGTGAGTTGGAGCAGAGAATCGCTACTTTGGAGTTCGACAGAGATAGAAAGTCCATGGGTGTCATGGTTGATTCTTCCTCCGGTAACAAGTTGTTGTTGGTCAAAGGAGCAGTTGAAAACGTTTTGGAGAGATCCACCCACATTCAATTGCTGGACGGTTCCAAGAGAGAATTGGACCAGTACTCCAGAGACTTGATCTTGCAGTCCTTGAGAGACATGTCCTTGTCCGCCTTGAGATGTTTGGGTTTCGCTTACTCTGACGTTCCATCCGATTTCGCTACTTACGATGGTTCTGAGGATCATCCAGCTCACCAACAGTTGCTGAACCCATCCAAACTACTCCTCCATCGAATCCAACCTGATCTTCGTTGGTTTCGTCGGTCTTAGAGACCCACCAAGAAAAGAAGTTAGACAGGCCATCGCTGATTGTAGAACCGCCGGTATCAGAGTTATGGTCATCACCGGAGATAACAAGTCCACTGCCGAGGCTATTTGTAGAGAGATCGGAGTTTTCGAGGCTGACGAGGACATTTCTTCCAGATCCCTGACCGGTATTGAGTTCATGGACGTCCAAGACCAGAAGAACCACTTGAGACAGACCGGTGGTTTGTTGTTCTCCAGAGCCGAACCAAGCACAAGCAAGAGATTGTCAGACTGCTGAAAGAGGACGGAGAAGTTGTTGCTATGACCGGTGATGGTGTTAATGACGCCCCAGCTTTGAAGTTGGCTGACATCGGTGTTGCTATGGGAATTTCCGGTACTGAAGTTGCTAAGGAAGCCTCCGATATGGTTTTGGCTGACGACAACTTTTCAACTATCGTTGCTGCTGTCGGAGAAGGTAGAAGTATCTACAACAACATGAAAGCCTTTATCAGATACATGATTTCCTCCAACATCGGTGAAGTTGCCTCCATTTTCTTGACTGCTGCCTTGGGTATTCCTGAGGGAATGATCCCAGTTCAGTTGTTGTGGGTTAACTTGGTTACTGACGGTCCACCTGCTACTGCTTTGGGTTTCAACCCACCAGACAAAGACATTATGAAGAGCCACCAAGAAGATCCGACGATTCCTTGATCACCGCCTGGATCTTGTTCAGATACATGGTCATCGGTCTTTATGTTGGTGTTGCCACCGTCGGTGTTTTCATCAATCTGGTACACCCACTCTTCCTTCATGGGTATTGACTTGTCTCAAGATGGTCATTCTTTGGTTTCCTACTCCAATTGGCTCATTGGGGACAATGTTCTTCCTGGGAGGGTTTCAAGGTTTCCCATTCACTGCTGGTTCCCAGACTTTCTCCTTCGATTCCAACCCATGTGACTACTTCCAGCAGGGAAAGATCAAGGCTTCCACCTTGTCTTTGTCCGTTTTGGTCGCCATTGAGATGTTCAACTCCCTGAACGCTTTGTCTGAGGACGGTTCCTTGGTTACTATGCCACCTTGGGTGAACCCATGGTTGTTGTTGGCTATGGCTGTTTCCTTCGGATTGCACTTCGTCATCCTGTACGTTCCATTCTTGGCCCAGGTTTTCGGTATTGTTCCACTGTCCTTGAACGAGTGGTTGTTGGTCTTGGCCGTTTCTTTGCCAGTTATCCTGATCGACGAGGTTTTGAAGTTCGTTGGTAGATGCACCTCTGGTTACAGATACTCCCCAAGAACTCGTCCACCAAGCAGAAAGAAGAGTAA 63 AtECA1MGKGSEDLVKKESLNSTPVNSDTFPAWAKDVAECEEHFVVSREKGLSSDEVLKRHQIYGLNELEKPEGTSIFKLILEQFNDTLVRILLAAAVISFVLAFFDGDEGGEMGITAFVEPLVIFLILIVNAIVGIWQETNAEKALEALKEIQSQQATVMRDGTKVSSLPAKELVPGDIVELRVGDKVPADMRVVALISSTLRVEQGSLTGESEAVSKTTKHVDENADIQGKKCMVFAGTTVVNGNCICLVTDTGMNTEIGRVHSQIQEAAQHEEDTPLKKKLNEFGEVLTMIIGLICALVWLINVKYFLSWEYVDGWPRNFKFSFEKCTYYFEIAVALAVAAIPEGLPAVITTCLALGTRKMAQKNALVRKLPSVETLGCTTVICSDKTGTLTTNQMAVSKLVAMGSRIGTLRSFNVEGTSFDPRDGKIEDWPMGRMDANLQMIAKIAAICNDANVEQSDQQFVSRGMPTEAALKVLVEKMGFPEGLNEASSDGDVLRCCRLWSELEQRIATLEFDRDRKSMGVMVDSSSGNKLLLVKGAVENVLERSTHIQLLDGSKRELDQYSRDLILQSLRDMSLSALRCLGFAYSDVPSDFATYDGSEDHPAHQQLLNPSNYSSIESNLIFVGFVGLRDPPRKEVRQAIADCRTAGIRVMVITGDNKSTAEAICREIGVFEADEDISSRSLTGIEFMDVQDQKNHLRQTGGLLFSRAEPKHKQEIVRLLKEDGEVVAMTGDGVNDAPALKLADIGVAMGISGTEVAKEASDMVLADDNFSTIVAAVGEGRSIYNNMKAFIRYMISSNIGEVASIFLTAALGIPEGMIPVQLLWVNLVTDGPPATALGFNPPDKDIMKKPPRRSDDSLITAWILFRYMVIGLYVGVATVGVFIIWYTHSSFMGIDLSQDGHSLVSYSQLAHWGQCSSWEGFKVSPFTAGSQTFSFDSNPCDYFQQGKIKASTLSLSVLVAIEMFNSLNALSEDGSLVTMPPWVNPWLLLAMAVSFGLHFVILYVPFLAQVFGIVPLSLNEWLL VLAVSLPVILIDEVLKFVGRCTSGYRYSPRTLSTKQKEE 64 PpPMR1/UPGAATTCATGACAGCTAATGAAAATCCTTTTGAGAATGAG 65 PpPMR1/LPGGCCGGCCTCAAACAGCCATGCTGTATCCATTGTATG 66 5′AOX1GCGACTGGTTCCAATTGACAAGCTT 67 PpPMR1/cLP GGTTGCTCTCGTCGATACTCAAGTGGGAAG68 AtECA1 /cLP GTCGGCTGGAACCTTATCACCAACTCTCAG 69 HumanATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATC calreticulinCTCCGCATTAGCTTACCCATACGACGTCCCAGACTACGCTTAC (hCRT)-DNACCATACGACGTCCCAGACTACGCTGAGCCCGCCGTCTACTTCAAGGAGCAGTTTCTGGACGGAGACGGGTGGACTTCCCGCTGGATCGAATCCAAACACAAGTCAGATTTTGGCAAATTCGTTCTCAGTTCCGGAAGTTCTACGGTGACGAGGAGAAAGATAAAGGTTTGCAGACAAGCCAGGATGCACGCTTTTATGCTCTGTCGGCCAGTTTCGAGCCTTTCAGCAACAAAGGCCAGACGCTGGTGGTGCAGTTCACGGTGAAACATGAGCAGAACATCGACTGTGGGGGCGGCTATGTGAAGCTGTTTCCTAATAGTTTGGACCAGACAGACATGCACGGAGACTCAGAATACAACATCATGTTTGGTCCCGACATCTGTGGCCCTGGCACCAAGAAGGTTCATGTCATCTTCAACTACAAGGGCAAGAACGTGCTGATCAACAAGGACATCCGTTGCAAGGATGATGAGTTTACACACCTGTACACACTGATTGTGCGGCCAGACAACACCTATGAGGTGAAGATTGACAACAGCCAGGTGGAGTCCGGCTCCTTGGAAGACGATTGGGACTTCCTGCCACCCAAGAAGATAAAGGATCCTGATGCTTCAAAACCGGAAGACTGGGATGAGCGGGCCAAGATCGATGATCCCACAGACTCCAAGCCTGAGGACTGGGACAAGCCCGAGCATATCCCTGACCCTGATGCTAAGAAGCCCGAGGACTGGGATGAAGAGATGGACGGAGAGTGGGAACCCCCAGTGATTCAGAACCCTGAGTACAAGGGTGAGTGGAAGCCCCGGCAGATCGACAACCCAGATTACAAGGGCACTTGGATCCACCCAGAAATTGACAACCCCGAGTATTCTCCCGATCCCAGTATCTATGCCTATGATAACTTTGGCGTGCTGGGCCTGGACCTCTGGCAGGTCAAGTCTGGCACCATCTTTGACAACTTCCTCATCACCAACGATGAGGCATACGCTGAGGAGTTTGGCAACGAGACGTGGGGCGTAACAAAGGCAGCAGAGAAACAAATGAAGGACAAACAGGACGAGGAGCAGAGGCTTAAGGAGGAGGAAGAAGACAAGAAACGCAAAGAGGAGGAGGAGGCAGAGGACAAGGAGGATGATGAGGACAAAGATGAGGATGAGGAGGATGAGGAGGACAAGGAGGAAGATGAGGAGGAAGATGTCCCCGGCCAGGCCCATGAC GAGCTGTAG 70 HumanMRFPSIFTAVLFAASSALAYPYDVPDYAYPYDVPDYAEPAVYFK calreticulinEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQ (hCRT)-TSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVK proteinLFPNSLDQTDMHGDSEYNIMFGPDICGPGTKKVHVIENYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPFKKIKDPDASKPEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPGQAHDEL 71 Human ERp57ATGCAATTCAACTGGAACATCAAGACTGTTGCTTCCATCTTGT (DNA)CCGCTTTGACTTTGGCTCAAGCTTCTGACGTTTTGGAGTTGACTGACGACAACTTCGAGTCCAGAATTTCTGACACTGGTTCCGCTGGATTGATGTTGGTTGAGTTCTTCGCTCCATGGTGTGGTCATTGTAAGAGATTGGCTCCAGAATACGAAGCTGCTGCTACTAGATTGAAGGGTATCGTTCCATTGGCTAAGGTTGACTGTACTGCTAACACTAACACTTGTAACAAGTACGGTGTTTCCGGTTACCCAACTTTGAAGATCTTCAGAGATGGTGAAGAAGCTGGAGCTTACGACGGTCCAAGAACTGCTGACGGTATCGTTTCCCACTTGAAGAAGCAAAGCTGGTCCAGCTTCTGTTCCATTGAGAACTGAGGAGGAGTTCAAGAAGTTCATCTCCGACAAGGACGCTTCTATCGTTGGTTTCTTCGACGATTCTTTCTCTGAAGCTCACTCCGAATTCTTGAAGGCTGCTTCCAACTTGAGAGACAACTACAGATTCGCTCACACTAACGTTGAGTCCCTTGGTTAACGAGTACGACGATAACGGTGAAGGTATCATCTTGTTCAGACCATCCCACTTGACTAACAAGTTCGAGGACAAGACAGTTGCTTACACTGAGCAGAAGATGACTTCCGGAAAGATCAAGAAGTTTATCCAAGAGAACATCTTCGGTATCTGTCCACACATGACTGAGGACAACAAGGACTTGATTCAGGGAAAGGACTTGTTGATCGCTTACTACGACGTTGACTACGAGAAGAACGCTAAGGGTTCCAACTACTGGAGAAACAGAGTTATGATGGTTGCTAAGAAGTTCTTGGACGCTGGTCACAAGTTGAACTTCGCTGTTGCTTCTAGAAAGACTTTCTCCCACGAGTTGTCTGATTTCGGATTGGAATCCACTGCTGGAGAGATTCCAGTTGTTGCTATCAGAACTGCTAAGGGAGAGAAGTTCGTTATGCAAGAGGAGTTCTCCAGAGATGGAAAGGCTTTGGAGAGATTCTTGCAGGATTACTTCGACGGTAACTTGAAGAGATACTTGAAGTCCGAGCCAATTCCAGAATCTAACGACGGTCCAGTTAAAGTTGTTGTTGCTGAGAACTTCGACGAGATCGTTAACAACGAGAACAAGGACGTTTTGATCGAGTTTTACGCTCCTTGGTGTGGACACTGTAAAAACTTGGAGCCAAAGTACAAGGAATTGGGTGAAAAGTTGTCCAAGGACCCAAACATCGTTATCGCTAAGATGGACGCTACTGCTAACGATGTTCCATCCCCATACGAAGTTAGAGGTTTCCCAACTATCTACTTCTCCCCAGCTAACAAGAAGTTGAACCCAAAGAAGTACGAGGGAGGTAGAGAATTGTCCGACTTCATCTCCTACTTGCAGAGAGAGGCTACTAATCCACCAGTTATCCAAGAGGAGAAGCCAAAGAAG AAGAAGAAAGCTCACGACGAGTTGTAG72 Human ERp57 MQFNWNIKTVASILSALTLAQASDVLELTDDNFESRISDTGSAGL (protein)MLVEFFAPWCGHCKRLAPEYEAAATRLKGIVPLAKVDCTANTNTCNKYGVSGYPTLKIFRDGEEAGAYDGPRTADGIVSHLKKQAGPASVPLRTEEEFKKFISDKDASIVGFFDDSFSEAHSEFLKAASNLRDNYRFAHTNVESLVNEYDDNGEGIILFRPSHLTNKFEDKTVAYTEQKMTSGKIKKFIQENIFGICPHMTEDNKDLIQGKDLLIAYYDVDYEKNAKGSNYWRNRVMMVAKKFLDAGHKLNFAVASRKTFSHELSDFGLESTAGEIPVVAIRTAKGEKFVMQEEFSRDGKALERFLQDYFDGNLKRYLKSEPIPESNDGPVKVVVAENFDEIVNNENKDVLIEFYAPWCGHCKNLEPKYKELGEKLSKDPNIVIAKMDATANDVPSPYEVRGFPTIYFSPANKKLNPKKYEGGRELSDFISYLQREATNPPVIQE EKPKKKKKAHDEL 73 hCRT-GTATACCCATACGACGTCCAGACTACGCTGAGCCCGCCGTCT BstZ17I- ACTTCAAGGAGC HA/UP74 hCRT-PacI/LP TTAATTAACTACAGCTCGTCATGGGCCTGGCCGGGGACATCTT CC 75Synthetic KLGFFKR peptide that binds CRT 76 hERdj3ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATC (DNA)CTCCGCATTAGCTGGTAGAGACTTCTACAAGATTTTGGGTGTTCCAAGATCCGCTTCCATCAAGGACATCAAGAAGGCTTACAGAAAGTTGGCTTTGCAATTGCACCCAGACAGAAACCCAGATGACCCACAAGCTCAAGAGAAGTTCCAAGACTTGGGTGCTGCTTACGAAGTTTTGTCCGATTCCGAGAAGAGAAAGCAGTACGACACTTACGGTGAAGAAGGATTGAAGGACGGTCACCAATCTTCTCACGGTGACATCTTCTCCCACTTTTTCGGTGACTTCGGTTTCATGTTCGGTGGTACTCCAAGACAACAGGACAGAAACATCCCAAGAGGTTCCGACATTATCGTTGACTTGGAGGTTACATTGGAAGAGGTTTACGCTGGTAACTTCGTTGAAGTTGTTAGAAACAAGCCAGTTGCTAGACAAGCTCCAGGTAAAGAAAGTGTAACTGTAGACAAGAGATGAGAACTACTCAGTTGGGTCCTGGTAGATTCCAAATGACACAGGAAGTTGTTTGCGACGAGTGTCCAAACGTTAAGTTGGTTAACGAAGAGAGAACTTTGGAGGTTGAGATCGAGCCAGGTGTTAGAGATGGAATGGAATACCCATTCATCGGTGAAGGTGAACCACATGTTGATGGTGAACCTGGTGACTTGAGATTCAGAATCAAAGTTGTTAAGCACCCAATCTTCGAGAGAAGAGGTGACGACTTGTACACTAACGTTACTATTTCCTTGGTTGAATCCTTGGTTGGTTTCGAGATGGACATCACTCATTTGAACGGTCACAAGGTTCACATTTCCAGAGACAAGATCACTAGACCAGGTGCTAAGTTGTGGAAGAAGGGTGAAGGATTGCCAAACTTCGACAACAACAACATCAAGGGATCTTTGATCATCACTTTCGACGTTGACTTCCCAAAAGAGCAGTTGACTGAAGAAGCTAGAGAGGGTATCAAGCAGTTGTTGAAGCAAGGTTCCGTTCAGAAGGTTTACAACGGATTGCAG GGATACTAA 77 hERdj3MRFPSIFTAVLFAASSALAGRDFYkiLGVPRSASIKDIKKAYRKLA (protein)LQLHPDRNPDDPQAQEKFQDLGAAYEVLSDSEKRKQYDTYGEEGLKDGHQSSHGDIFSHFFGDFGFMFGGTPRQQDRNIPRGSDIIVDLEVTLEEVYAGNFVEVVRNKPVARQAPGKRKCNCRQEMRTTQLGPGRFQMTQEVVCDECPNVKLVNEERTLEVEIEPGVRDGMEYPFIGEGEPHVDGEPGDLRFRIKVVKHPIFERRGDDLYTNVTISLVESLVGFEMDITHLDGHKVHISRDKITRPGAKLWKKGEGLPNFDNNNIKGSLIITFDVDFPKEQLTEEAREGIKQLLKQGSVQKVYNGLQGY

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1. A lower eukaryote host cell in which the function of at least oneendogenous gene encoding a chaperone protein has been disrupted ordeleted and a nucleic acid molecule encoding at least one mammalianhomolog of the endogenous chaperone protein is expressed in the hostcell.
 2. The lower eukaryote host cell of claim 1, wherein the chaperoneprotein is a Protein Disulphide Isomerase (PDI).
 3. The lower eukaryotehost cell of claim 1, wherein the mammalian homolog is a human PDI. 4.The lower eukaryote host cell of claim 1, wherein the host cell furtherincludes a nucleic acid molecule encoding a recombinant protein.
 5. Thelower eukaryote host cell of claim 1, wherein the function of at leastone endogenous gene encoding a protein O-mannosyltransferase (PMT)protein has been reduced, disrupted, or deleted.
 6. The lower eukaryotehost cell of claim 1, wherein the host cell further includes a nucleicacid molecule encoding an endogenous or heterologous Ca²⁺ ATPase.
 7. Thelower eukaryote host cell of claim 1, wherein the host cell furtherincludes a nucleic acid molecule encoding an ERp57 protein and a nucleicacid molecule encoding a calreticulin protein. 8-13. (canceled)
 14. Amethod for producing a recombinant protein comprising: (a) providing alower eukaryote host cell in which the function of at least oneendogenous gene encoding a chaperone protein has been disrupted ordeleted and a nucleic acid molecule encoding at least one mammalianhomolog of the endogenous chaperone protein is expressed in the hostcell; (b) introducing a nucleic acid molecule into the host cellencoding the recombinant protein: and (c) growing the host cell underconditions suitable for producing the recombinant protein.
 15. Themethod of claim 14, wherein the chaperone protein is a ProteinDisulphide Isomerase (PDI) and the mammalian homolog is a human PDI. 16.(canceled)
 17. The method of claim 14, wherein the function of at leastone endogenous gene encoding a protein O-mannosyltransferase (PMT)protein has been reduced, disrupted, or deleted.
 18. The method of claim14, wherein the host cell further includes a nucleic acid moleculeencoding an endogenous or heterologous Ca²⁺ ATPase.
 19. The method ofclaim 14, wherein the host cell further includes a nucleic acid moleculeencoding an ERp57 protein and a nucleic acid molecule encoding acalreticulin protein.
 20. A method for producing a recombinant proteinhaving reduced O-glycosylation comprising: (a) providing a lowereukaryote host cell in which the function of at least one endogenousgene encoding a chaperone protein has been disrupted or deleted and anucleic acid molecule encoding at least one mammalian homolog of theendogenous chaperone protein is expressed in the host cell; (b)introducing a nucleic acid molecule into the host cell encoding therecombinant protein: and (c) growing the host cell under conditionssuitable for producing the recombinant protein.
 21. The method of claim20, wherein the chaperone protein is a Protein Disulphide Isomerase(PDI) and the mammalian homolog is a human PDI.
 22. (canceled)
 23. Themethod of claim 20, wherein the function of at least one endogenous geneencoding a protein O-mannosyltransferase (PMT) protein has been reduced,disrupted, or deleted.
 24. The method of claim 20, wherein the host cellfurther includes a nucleic acid molecule encoding an endogenous orheterologous Ca2+ ATPase.
 25. The method of claim 20, wherein the hostcell further includes a nucleic acid molecule encoding an ERp57 proteinand a nucleic acid molecule encoding a calreticulin protein.
 26. Themethod of claim 20, wherein the recombinant protein is selected from thegroup consisting of mammalian or human enzymes, cytokines, growthfactors, hormones, vaccines, antibodies, and fusion proteins.
 27. Themethod of claim 14, wherein the recombinant protein is selected from thegroup consisting of mammalian or human enzymes, cytokines, growthfactors, hormones, vaccines, antibodies, and fusion proteins.
 28. Thelower eukaryote host cell of claim 1, wherein the recombinant protein isselected from the group consisting of mammalian or human enzymes,cytokines, growth factors, hormones, vaccines, antibodies, and fusionproteins.